Electrode array device having an adsorbed porous reaction layer

ABSTRACT

There is disclosed an electrode array device having an adsorbed porous reaction layer for improved synthesis quality. The array comprises a plurality of electrodes on a substrate, wherein the electrodes are electronically connected to a computer control system. The array has an adsorbed porous reaction layer on the plurality of electrodes, wherein the adsorbed porous reaction layer comprises a chemical species having at least one hydroxyl group. In the preferred embodiment, the reaction layer is sucrose. A method for preparing an electrode array for improved synthesis quality is disclosed. The method comprises a cleaning method and a method of attachment of a reaction layer. The cleaning method comprises a plasma cleaning method and a chemical cleaning method. The reaction layer is attached after cleaning by exposing the microarray to a solution containing the chemical species having at least one hydroxyl group.

PRIORITY CLAIM

The present application is a continuation of U.S. patent applicationSer. No. 15/155,046, entitled “ELECTRODE ARRAY DEVICE HAVING AN ADSORBEDPOROUS REACTION LAYER” filed May 15, 2016, which is a continuationapplication of U.S. patent application Ser. No. 14/082,971 entitled“ELECTRODE ARRAY DEVICE HAVING AN ADSORBED POROUS REACTION LAYER” filedNov. 18, 2013 which issued May 17, 2016 as U.S. Pat. No. 9,339,782 andwhich is a continuation of U.S. patent application Ser. No. 13/571,306entitled “ELECTRODE ARRAY DEVICE HAVING AN ADSORBED POROUS REACTIONLAYER” filed Aug. 9, 2012, abandoned Jun. 11, 2014, which is acontinuation of U.S. patent application Ser. No. 11/863,097 entitled“ELECTRODE ARRAY DEVICE HAVING AN ADSORBED POROUS REACTION LAYER” filedSep. 27, 2007, abandoned Oct. 1, 2012, which is a continuation of U.S.patent application Ser. No. 10/992,252 entitled “ELECTRODE ARRAY DEVICEHAVING AN ADSORBED POROUS REACTION LAYER” inventors Maurer et al., filedNov. 18, 2004, abandoned Feb. 1, 2011.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file CUST-01113US4_ST25.TXT, createdNov. 22, 2017, 1011 bytes, machine format IBM-PC, MS-Windows operatingsystem, is hereby incorporated by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention provides an electrode microarray having anadsorbed porous reaction layer for improved synthesis quality.Specifically, the present invention provides an electrode microarrayhaving a plurality of electrodes, having an adsorbed porous reactionlayer and a process for treating the microarray surface to significantlyenhance nucleic acid synthesis quality and increase assay sensitivity.More specifically, the present invention provides a computer controlledelectrode microarray having a plurality of platinum-containingelectrodes, having an adsorbed porous reaction layer and a process oftreating the microarray surface to significantly enhance the quality ofsynthesis of oligonucleotides, peptides, and other chemical specieswhile increasing binding assay sensitivity.

BACKGROUND OF THE INVENTION

Microarrays and particularly nucleic acid microarrays have becomeimportant analytical research tools in pharmacological and biochemicalresearch and discovery. Microarrays are miniaturized arrays of points orlocations arranged in a column and row format. Molecules, includingbiomolecules, are attached or synthesized in situ at specific attachmentpoints, which are usually in a column and row format although otherformats may be used. An advantage of microarrays is that they providethe ability to conduct hundreds, if not thousands, of experiments inparallel. Such parallelism, as compared to sequential experimentation,can be used to increase the efficiency of exploring relationshipsbetween molecular structure and biological function, where slightvariations in chemical structure can have profound biochemical effects.Microarrays are available in different formats and have differentsurface chemistry characteristics that lead to different approaches forattaching or synthesizing molecules. Differences in microarray surfacechemistry lead to differences in preparation methods for providing asurface that is receptive to attachment of a presynthesized chemicalspecies or for synthesizing a chemical species in situ. As the namesuggests, the attachment points on microarrays are of a micrometerscale, which is generally 1-100 μm.

Research using microarrays has focused mainly on deoxyribonucleic acid(DNA) and ribonucleic acid (RNA) related areas, which includes genomics,cellular gene expression, single nucleotide polymorphisms (SNP), genomicDNA detection and validation, functional genomics, and proteomics(Wilgenbus and Lichter, J. Mol. Med. 77:761, 1999; Ashfari et al.,Cancer Res. 59:4759, 1999; Kurian et al., J. Pathol. 187:267, 1999;Hacia, Nature Genetics 21 suppl.:42, 1999; Hacia et al., Mol. Psychiatry3:483, 1998; and Johnson, Curr. Biol. 26:R171, 1998.) In addition tomicroarrays for DNA/RNA research, microarrays can be used for researchrelated to peptides (two or more linked natural or synthetic aminoacids), small molecules (such as pharmaceutical compounds), oligomers,and polymers.

Considering microarrays for DNA related research, there are numerousmethods for preparing a microarray of DNA related molecules. DNA relatedmolecules include native or cloned DNA and synthetic DNA. Syntheticrelatively short single-stranded DNA or RNA strands are commonlyreferred to as oligonucleotides (oligos), which is synonymous witholigodeoxyribonucleotide. Microarray preparation methods include thefollowing: (1) spotting a solution on a prepared flat surface usingspotting robots; (2) in situ synthesis by printing reagents via ink jetor other printing technology and using regular phosphoramiditechemistry; (3) in situ parallel synthesis using electrochemicallygenerated acid for deprotection and using regular phosphoramiditechemistry; (4) maskless photo-generated acid (PGA) controlled in situsynthesis and using regular phosphoramidite chemistry; (5) mask-directedin situ parallel synthesis using photo-cleavage of photolabileprotecting groups (PLPG); (6) maskless in situ parallel synthesis usingPLPG and digital photolithography; and (7) electric fieldattraction/repulsion for depositing oligos.

Photolithographic techniques for in situ olio synthesis are disclosed inFodor et al. U.S. Pat. No. 5,445,934 and the additional patents claimingpriority thereto. Electric field attraction/repulsion microarrays aredisclosed in Hollis et al. U.S. Pat. No. 5,653,939 and Heller et al.U.S. Pat. No. 5,929,208. An electrode microarray for in situ oligosynthesis using electrochemical deblocking is disclosed in MontgomeryU.S. Pat. Nos. 6,093,302; 6,280,595, and 6,444,111 (Montgomery I, II,and III respectively), which are incorporated by reference herein.Another and materially different electrode array (not a microarray) forin situ oligo synthesis on surfaces separate and apart from electrodesusing electrochemical deblocking is disclosed in Southern U.S. Pat. No.5,667,667. A review of oligo microarray synthesis is provided by Gao etal., Biopolymers 2004, 73, 579.

Microarrays other than DNA microarrays have been disclosed. For example,the synthetic preparation of a peptide array was originally reported in1991 using photolithography masking techniques. This method was extendedin year 2000 to include an addressable masking technique usingphotogenerated acids and/or in combination with photosensitizers fordeblocking. Reviews of peptide microarray synthesis using photolabiledeblocking are provided by Pellois et al., J. Comb. Chem. 2000, 2:355and Fodor et al., Science, 1991, 251:767. Recent work using peptidearrays has utilized arrays produced by spotting pre-synthesized peptidesor isolated proteins. A review of protein arrays is provided by Cahilland Nordhoff, Adv. Biochem. Engin/Biotechnol. 2003, 83:177.

Preparation methods for providing a microarray surface that is receptiveto attachment of a presynthesized chemical species or for synthesizing achemical species in situ must provide a surface that is capable ofbonding a chemical species as well as being capable of providing thechemical functionality necessary to conduct pharmacological andbiochemical research and discovery. One approach is to treat a surfaceto provide reactive groups capable of covalently bonding to chemicalspecies of interest. In such an approach, the reactive group istypically present as a result of a surface treatment or coating of thesurface. For DNA related species, the reactive group required is ahydroxyl, unless there has been a chemical modification. For peptides,the reactive group required is an amine, unless there has been achemical modification.

Glass is a commonly used solid substrate for microarrays and must betreated before use. A common glass treatment uses silanization chemistryto introduce a stable and uniform surface having reactive groups forattachment or in situ synthesis of oligos or other chemical species (Guoet al., 1994, Nucl. Acids Res., 22:5456-5465; LeProust et al., 2001,Nucl. Acids Res., 29:2171-2180; Maskos and Southern, 1992, Nucl. AcidsRes., 20:1679-1684; Skrzypcznski et al., U.S. Patent Appl. Pub.2004/0073017, and Southern et al. U.S. Pat. No. 6,576,426.). Glass beadsfor bulk synthesis must also undergo silanization (Maskos and Southern).Gold surfaces are treated with thiol linker chemistry (Kelley et al.U.S. Patent Pub. 2002/0172963). Similarly, polymeric microarraysupports, such as polypropylene, must be treated by oxidation followedby introduction of reactive groups such as terminal amines (Schepinov etal., 1997, Nucl. Acids Res., 25:1155-1161). Additionally, polystyrenebeads are surface treated with polyethylene glycols having reactiveterminal groups for bulk synthesis of peptides (Merck, Inc. NovabiochemDiv. and Aldrich et al., U.S. Patent Appl. Pub. 2003/0134989). Finally,the surface of electrodes on an electrode microarray must be treatedwith a surface coating to provide reactive groups (Montgomery I, II, andIII). For oligo synthesis, such a surface coating must be able towithstand the rigors of repeated exposure to synthesis solutions and toelectrochemical deblocking solutions.

The electrochemical synthesis microarray disclosed in Montgomery I, II,and III is based upon a semiconductor chip having a plurality ofmicroelectrodes in a column and row format. This chip design usesComplimentary Metal Oxide Semiconductor (CMOS) technology to createhigh-density arrays of microelectrodes with parallel addressing forselecting and controlling individual microelectrodes within the array.The electrodes are “turned on” by applying a voltage, which generateselectrochemical reagents (particularly acidic protons) that alter the pHin a small, defined “virtual flask” region or volume adjacent to theelectrode. In order to provide reactive groups at each electrode, themicroarray is coated with a porous matrix material. Biomolecules can besynthesized at any of the electrodes, and such synthesis occurs withinthe porous matrix material. For the deblocking step, the pH iselectrochemically decreased by applying a voltage to an electrode. ThepH decreases only in the vicinity of the electrode because the abilityof the acidic reagent to travel away from an electrode is limited bynatural diffusion and by a buffer in solution.

In general, when a surface is treated, there is a reactive group at theterminal end of a linker that is attached to the surface duringtreatment. A linker is a molecule that connects a species of interest toa solid surface. For example, a linker for glass has a reactive group atone end and a silane-coupling group at the other for bonding to glass.Linkers can be of various lengths, depending on the particular chemicalspecies used to form the linker. In addition to linkers, spacers can beattached to a linker in order to provide more distance between a solidsurface and an attached chemical species. Spacers can be of a differentchemistry than linkers. Linkers and spacers for attachment of oligos aredisclosed for glass supports (Guo et al., LeProus et. al., Maskos etal., Skrzypcznski and Southern) for aminated polypropylene supports(Schepinov et al.) and for polystyrene beads (Merck, Inc. NovabiochemDiv. and Aldrich et al.).

For a surface coating on an electrode microarray, the surface coatingitself provides reactive groups that are naturally present within thecoating. Montgomery I, II, and III disclose a surface coating comprisingcontrolled porosity glass (CPG); generic polymers, such as, teflons,nylons, polycarbonates, polystyrenes, polyacylates, polycyanoacrylates,polyvinyl alcohols, polyamides, polyimides, polysiloxanes,polysilicones, polynitriles, polyelectrolytes, hydrogels, expoxypolymers, melamines, urethanes and copolymers and mixtures of these andother polymers; biologically derived polymers, polyhyaluric acids,celluloses, and chitons; ceramics, such as, alumina, metal oxides,clays, and zeolites; surfactants; thiols; self-assembled monolayers;porous carbon; and fullerine materials. Montgomery I, II, and IIIfurther discloses that the surface coating can be attached to theelectrodes by spin coating, dip coating or manual application, or anyother acceptable form of coating. Montgomery I, II, and III furtherdiscloses linker molecules attached to controlled porosity glass viasilicon-carbon bonds and that the linker molecules include aryl,acetylene, ethylene glycol oligomers containing from 2 to 10 monomerunits, diamines, diacids, amino acids, and combinations thereof. In eachinstance, Montgomery discloses coating the entire surface of amicroarray device and not just electrode surfaces.

Guo et al. discloses the use of a 23-atom linker for covalentlyattaching a DNA sequence to glass. The linker is made by reaction of theglass surface with aminopropyltrimethoxysilane to provide anamino-derivatized surface followed by coupling of the amino groups withexcess p-phenylenediisothiocyanate to convert the amino groups toamino-reactive phenylisothiocyanate groups. An oligonucleotide is thencovalently attached to the amino-reactive group by coupling to theamino-reactive group a 5′ amino-modified oligonucleotide attached to the5′ end of a sequence of an oligonucleotide. The resulting structure is asolid surface having a linker attached thereto and the linker having anoligonucleotide attached from the 5′ side to the linker. Guo et al.further disclose a spacer comprising up to a 15-deoxythymidylate chainthat is between the oligonucleotide and the linker. The spacer has a 5′amino-modified oligonucleotide to allow attachment to the amino-reactivegroup. The spacer is attached onto the 5′ end of an oligonucleotide as apart of the oligonucleotide, and then the spacer-oligonucleotide isattached to the linker. As viewed from the glass surface, the finalstructure provides a glass surface having a linker having attachedthereto a 5′ to 3′ prime spacer-oligonucleotide, where thespacer-oligonucleotide has been synthesized elsewhere and then attachedto the linker. The 15-deoxythymidylate chain was found to have thehighest hybridization signal compared to chains having fewerdeoxythymidylate units.

Maskos and Southern disclose silane-coupled linkers for glass. Thelinkers are different length and are terminated with a hydroxyl foroligonucleotide synthesis on the glass. The linkers are bound to glassthrough a glycidoxypropyl silane linkage and have a hexaethylene glycolmiddle section of different lengths. The linkers range from 8 to 26atoms in length and do not have any charge. Shchepinov et al. disclosesspacer molecules for coupling oligonucleotides to aminatedpolypropylene. The spacer molecules are built using phosphoramiditechemistry and synthesized monomers having diols as a part of themonomeric unit. Both 3′ and 5′ oligonucleotides were built upon thespacers.

LeProust et al. discloses silane linkers terminating in a hydroxyl,amide, or amine group. The linkers were used to synthesizeoligonucleotides (deoxythymidylate units) on glass slides to determinethe efficiency/fidelity of synthesis. The linkers were nonionic.Southern et al. discloses nonionic linkers/spacers for use on controlpore glass (CPG) for oligonucleotide synthesis. The linkers wereattached to CPG through a terminal amine attached to a group on the CPGvia silanization. Skrzypcznski et al. discloses nonionic linkers/spacercoupled to glass or sol-gel glass coating through silane linkage. Thelinker/spacer is proposed to have a hydrophobic part next to the glassattached to a hydrophilic part where a DNA probe is attached.

Linkers and spacers are sometimes used for peptide synthesis off of amicroarray. Specifically, microscopic polystyrene (PS) beads are used asa solid support (Aldrich et al.). The beads have a polyethylene glycol(PEG) spacer attached to the beads and a linking group attached to thePEG, where the linking group has a reactive group for synthesis ofpeptides. After synthesis, the peptides are cleaved from the linkinggroup and recovered. Numerous PS-PEG resins for synthesis are availablecommercially from Merck Company, Novabiochem Division, as well as othersources.

Oligo microarrays made with the electrochemical process as disclosed inMontgomery I, II, and III have had problems with oligo quality, wherequality is judged by missing deoxynucleotide bases in sequencesresulting from inefficient deblocking. In addition, quality problems canarise from delamination of the coating over the electrodes. Control poreglass coatings and polysaccharide agarose coatings are both prone todelamination quality problems. Such quality problems have caused theresulting oligo microarray to be less useful for sensitivity of geneexpression assays (i.e., finding low abundance mRNA species) and forsingle nucleotide polymorphisms (SNP) assays, wherein single basechanges need to be detected. Peptide synthesis on electrode microarrayshas also been problematic. Similar quality problems have been found forglass microarrays, where research has found inefficient reactions of thevarious reagents with functional groups close to glass plate surfaces(LeProust et al.).

Considering (1) the above discussion of electrode microarray qualityproblems for oligonucleotides, peptides, and other chemical species, and(2) the need for a surface having reactive groups on electrodemicroarrays, there is a need in the art to be able to improve in situelectrochemical synthesis quality to provide microarrays having higherquality. The present invention addresses these needs. Additionally, forelectrode microarrays, there is a need for a modified surface coatingincorporating a linker and spacer to improve synthesis quality andprevent fluorescence quenching.

SUMMARY OF THE INVENTION

The present invention provides an electrode microarray having anadsorbed porous reaction layer for improved synthesis quality. Themicroarray comprises a plurality of electrodes on a substrate, whereinthe electrodes are electronically connected to a computer controlsystem. In addition, the microarray has an adsorbed porous reactionlayer on the plurality of electrodes, wherein the adsorbed porousreaction layer comprises a chemical species having at least one hydroxylgroup. The chemical species is selected from the group consisting ofmonosaccharides, disaccharides, trisaccharides, polyethylene glycol,polyethylene glycol derivative, N-hydroxysuccinimide, formula I, formulaII, formula III, formula IV, formula V, formula VI, and formula VII, andcombinations thereof. Formula I is

formula II is

formula III is HOR⁴(OR⁵)_(m)R⁷, formula IV is

formula V is

formula VI is

and formula VII is

The subscript m is an integer from ranging from 1 to about 4. Thepolyethylene glycol has a molecular weight of approximately 1,000 to20,000 daltons.

R¹, R², R⁷, and R⁸ are independently selected from the group consistingof hydrogen, and substituted and unsubstituted alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, andpolycyclic group, and halo, amide, alkoxy, acyl, acyloxy, oxycarbonyl,acyloxycarbonyl, alkoxycarbonyloxy, carboxy, amino, secondary amino,tertiary amino, hydrazino, azido, alkazoxy, cyano, isocyano, cyanato,isocyanato, thiocyanato, fulminato, isothiocyanato, isoselenocyanato,selenocyanato, carboxyamido, acylimino, nitroso, aminooxy,carboximidoyl, hydrazonoyl, oxime, acylhydrazino, amidino, sulfide,sulfoxide, thiosulfoxide, sulfone, thiosulfone, sulfate, thiosulfate,hydroxyl, formyl, hydroxyperoxy, hydroperoxy, peroxy acid, carbamoyl,trimethyl silyl, nitro, nitroso, oxamoyl, pentazolyl, sulfamoyl,sulfenamoyl, sulfeno, sulfinamoyl, sulfino, sulfo, sulfoamino,hydrothiol, tetrazolyl, thiocarbamoyl, thiocarbazono, thiocarbodiazono,thiocarbonohydrazido, thiocarboxy, thioformyl, thioacyl, thiocyanato,thio semicarbazido, thio sulfino, thio sulfo, thioureido, triazano,triazeno, triazinyl, trithiosulfo, sulfinimidic acid, sulfonimidic acid,sulfinohydrazonic acid, sulfonohydrazonic acid, sulfinohydroximic acid,sulfonohydroximic acid, and phosphoric acid ester.

R³ is selected from the group consisting of heteroatom group, carbonyl,and substituted and unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclicgroup. R⁴ and R⁵ are independently selected from the group consisting ofmethylene, ethylene, propylene, butylene, pentylene, and hexylene. R⁶forms a ring structure with two carbons of succinimide and is selectedfrom the group consisting of substituted and unsubstituted alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heterocyclic ring, and polycyclic group. R⁷ is selected from the groupconsisting of amino and hydroxyl.

The monosaccharide is selected from the group consisting of allose,altrose, arabinose, deoxyribose, erythrose, fructose, galactose,glucose, gulose, idose, lyxose, mannose, psicose, L-rhamnose, ribose,ribulose, sedoheptulose, D-sorbitol, sorbose, sylulose, tagatose,talose, threose, xylulose, and xylose. The disaccharide is selected fromthe group consisting of amylose, cellobiose, lactose, maltose,melibiose, palatinose, sucrose, and trehalose. The triaccharide isselected from the group consisting of raffinose and melezitose.

The polyethylene glycol derivative is selected from the group consistingof diethylene glycol, tetraethylene glycol, polyethylene glycol havingprimary amino groups, 2-(2-aminoethoxy) ethanol, ethanol amine,di(ethylene glycol) mono allyl ether, di(ethylene glycol) mono tosylate,tri(ethylene glycol) mono allyl ether, tri(ethylene glycol) monotosylate, tri(ethylene glycol) mono benzyl ether, tri(ethylene glycol)mono trityl ether, tri(ethylene glycol) mono chloro mono methyl ether,tri(ethylene glycol) mono tosyl mono allyl ether, tri(ethylene glycol)mono allyl mono methyl ether, tetra(ethlyne glycol) mono allyl ether,tetra(ethylene glycol) mono methyl ether, tetra(ethylene glycol) monotosyl mono allyl ether, tetra(ethylene glycol) mono tosylate,tetra(ethylene glycol) mono benzyl ether, tetra(ethylene glycol) monotrityl ether, tetra(ethylene glycol) mono 1-hexenyl ether,tetra(ethylene glycol) mono 1-heptenyl ether, tetra(ethylene glycol)mono 1-octenyl ether, tetra(ethylene glycol) mono 1-decenyl ether,tetra(ethylene glycol) mono 1-undecenyl ether, penta(ethylene glycol)mono methyl ether, penta(ethylene glycol) mono allyl mono methyl ether,penta(ethylene glycol) mono tosyl mono methyl ether, penta(ethyleneglycol) mono tosyl mono allyl ether, hexa(ethylene glycol) mono allylether, hexa(ethylene glycol) mono methyl ether, hexa(ethylene glycol)mono benzyl ether, hexa(ethylene glycol) mono trityl ether,hexa(ethylene glycol) mono 1-hexenyl ether, hexa(ethylene glycol) mono1-heptenyl ether, hexa(ethylene glycol) mono 1-octenyl ether,hexa(ethylene glycol) mono 1-decenyl ether, hexa(ethylene glycol) mono1-undecenyl ether, hexa(ethylene glycol) mono 4-benzophenonyl mono1-undecenyl ether, hepta(ethylene glycol) mono allyl ether,hepta(ethylene glycol) mono methyl ether, hepta(ethylene glycol) monotosyl mono methyl ether, hepta(ethylene glycol) monoallyl mono methylether, octa(ethylene glycol) mono allyl ether, octa(ethylene glycol)mono tosylate, octa(ethylene glycol) mono tosyl mono allyl ether,undeca(ethylene glycol) mono methyl ether, undeca(ethylene glycol) monoallyl mono methyl ether, undeca(ethylene glycol) mono tosyl mono methylether, undeca(ethylene glycol) mono allyl ether, octadeca(ethyleneglycol) mono allyl ether, octa(ethylene glycol), deca(ethylene glycol),dodeca(ethylene glycol), tetradeca(ethylene glycol), hexadeca(ethyleneglycol), octadeca(ethylene glycol), benzophenone-4-hexa(ethylene glycol)allyl ether, benzophenone-4-hexa(ethylene glycol) hexenyl ether,benzophenone-4-hexa(ethylene glycol) octenyl ether,benzophenone-4-hexa(ethylene glycol) decenyl ether,benzophenone-4-hexa(ethylene glycol) undecenyl ether,4-flourobenzophenone-4′-hexa(ethylene glycol) allyl ether,4-flourobenzophenone-4′-hexa(ethylene glycol) undecenyl ether,4-hydroxybenzophenone-4′-hexa(ethylene glycol) allyl ether,4-hydroxybenzophenone-4′-hexa(ethylene glycol) undecenyl ether,4-hydroxybenzophenone-4′-tetra(ethylene glycol) allyl ether,4-hydroxybenzophenone-4′-tetra(ethylene glycol) undecenyl ether,4-morpholinobenzophenone-4′-hexa(ethylene glycol) allyl ether,4-morpholinobenzophenone-4′-hexa(ethylene glycol) undecenyl ether,4-morpholinobenzophenone-4′-tetra(ethylene glycol) allyl ether, and4-morpholinobenzophenone-4′-tetra(ethylene glycol) undecenyl ether.

The present invention further provides a process for forming anelectrode array having an adsorbed porous reaction layer for improvedsynthesis quality. The process comprises (1) providing a plurality ofclean electrodes on a substrate, wherein the electrodes areelectronically connected to a computer control system; and (2) adsorbinga porous reaction layer on the plurality of electrodes, wherein theporous reaction layer comprises a chemical species having at least onehydroxyl group, wherein the chemical species is selected from the groupconsisting of monosaccharides, disaccharides, trisaccharides,polyethylene glycol, polyethylene glycol derivative,N-hydroxysuccinimide, formula I, formula II, formula III, formula IV,formula V,

The present invention further provides a process for adsorbing a porousreaction layer onto a plurality of electrodes, comprising (1) contactinga treatment solution to the microarray for from about 1 minute to about2 weeks, wherein the treatment solution comprises the chemical speciesthat adsorbs onto each electrode of the plurality of electrodes and asolvent capable of dissolving the chemical species; and (2) washing offthe treatment solution while leaving a layer of chemical speciesadsorbed onto each electrode of the plurality of electrodes.

The present invention further provides a process for cleaning anelectrode microarray comprising (1) etching the electrode microarraysurface using a plasma cleaning method; and (2) cleaning the electrodemicroarray using a chemical cleaning method. Preferably, the plasmacleaning method comprises exposing the electrode microarray to an argonplasma sputter etch process for approximately two to six minutes, wherethe plasma power is 200 W, the self bias voltage is 600-650V, the plasmapressure is 8 mTorr, and a 200 mm diameter electrode is used in aparallel plate plasma chamber. Preferably, the plasma cleaning methodcomprises exposing the electrode microarray to a sulfur hexafluorideplasma for approximately 30 to 60 minutes, where the plasma power is 300watts, the plasma pressure is approximately 250 to 350 mTorr, and thegas flow is 124 cubic centimeters per minute in an isoptropic plasmachamber. Preferably, the plasma cleaning method comprises etching theelectrode microarray in a commercial Reactive Ion Etch Plasma system(such as Oxford Plasmalab 800Plus RIE system with a 460 mm diameterelectrode) using (1) an argon plasma for approximately 2 to 4 minutesand a RF plasma power of approximately 600 watts, where the pressure isapproximately eight millitorr and the Ar gas flow is approximately 30sccm; (2) an oxygen plasma for approximately 5 to 7 minutes using apower of approximately 500 watts, where the pressure is approximately 50millitorr and the oxygen gas flow of approximately 50 sccm; or (3) anargon plasma for approximately 8 to 12 minutes using a power ofapproximately 600 watts, where the pressure is approximately eightmillitorr and the Ar gas flow is approximately 30 sccm.

Preferably, chemical cleaning method comprises an electrochemicalcleaning method comprising (1) contacting a sulfuric acid solution withthe electrodes of the electrode microarray, wherein the sulfuric acidsolution has a concentration of approximately 0.01 to 5 molar and theelectrode microarray is electronically attached to a control system; (2)pulsing a current for approximately 0.01 to 60 seconds to a first groupof electrodes while a second group of electrodes is grounded; (3)pulsing a current for approximately 0.01 to 60 seconds to the secondgroup of electrodes while the first group of electrodes is grounded; and(4) alternating between pulsing a current for approximately 0.01 to 60seconds to the first group of electrodes while the second group ofelectrodes is grounded and pulsing a current for approximately 0.01 to60 seconds to the second group of electrodes while the first group ofelectrodes remains grounded for a cumulative time of approximately 1 to60 minutes. Preferably, the chemical cleaning method comprises ahydrogen peroxide cleaning method comprising contacting a hydrogenperoxide solution with the electrodes of the electrode microarray,wherein the hydrogen peroxide solution has a concentration ofapproximately 0.5 to 10% (by volume), contacting time is approximately 1minute to 24 hours, and the hydrogen peroxide solution temperature isapproximately 20 to 95 degrees Celsius.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are schematics of a cross section of two electrodes of amicroarray of electrodes. In FIG. 1A, an adsorbed porous reaction layeris shown as being adsorbed across the entire microarray surface. In FIG.1B, an adsorbed porous reaction layer is shown as being adsorbedpredominately on only electrode surfaces. The reaction layer is shownhaving hydroxyl moieties as reactive groups.

FIGS. 2A and 2B are schematics of a cross section of two electrodes of amicroarray of electrodes, wherein there is a bound linker moiety has aterminal reactive group for in situ synthesis.

FIG. 3 is a schematic of a cross section of two electrodes of amicroarray of electrodes, wherein a comparison is made between alinker/spacer having no charge to one have charge. Sequence listing:Element 316 (TTTTTTTTTTTTTTTT) (SEQ ID NO:3).

FIG. 4 is a photograph of a magnified portion of a top view of amicroarray having agarose as a reaction layer. The lighter spots arefluorescence of fluorescently labeled nucleotides that were hybridizedwith the array after in situ synthesis of DNA oligos.

FIG. 5 is a photograph of a magnified portion of a top view of amicroarray having sucrose as a reaction layer. The lighter spots arefluorescence of fluorescently labeled nucleotides that were hybridizedwith the array after in situ synthesis of DNA oligos.

FIG. 6 is a bar chart displaying the results of a sensitivity studyusing sucrose as the reaction layer.

FIG. 7 is a photograph of a magnified portion of a top view of amicroarray having diethylene glycol as a reaction layer. The lighterspots are fluorescence of fluorescently labeled nucleotides that werehybridized with the array after in situ synthesis of DNA oligos.

FIG. 8 is a photograph of a magnified portion of a top view of amicroarray having ethylene glycol as a reaction layer. The lighter spotsare fluorescence of fluorescently labeled nucleotides that werehybridized with the array after in situ synthesis of DNA oligos.

FIG. 9 is a photograph of a magnified portion of a top view of amicroarray having N-hydroxysuccinimide as a reaction layer. The lighterspots are fluorescence of fluorescently labeled nucleotides that werehybridized with the array after in situ synthesis of DNA oligos.

FIG. 10 is a photograph of a magnified portion of a top view of amicroarray having triethylene glycol as a reaction layer. The lighterspots are fluorescence of fluorescently labeled nucleotides that werehybridized with the array after in situ synthesis of DNA oligos.

FIG. 11 is a photograph of a magnified portion of a top view of amicroarray having raffinose as a reaction layer. The lighter spots arefluorescence of fluorescently labeled nucleotides that were hybridizedwith the array after in situ synthesis of DNA oligos.

FIG. 12 is a photograph of a magnified portion of a top view of amicroarray having melizitose as a reaction layer. The lighter spots arefluorescence of fluorescently labeled nucleotides that were hybridizedwith the array after in situ synthesis of DNA oligos.

FIG. 13 is a photograph of a magnified portion of a top view of amicroarray having Splenda® as a reaction layer. The lighter spots arefluorescence of fluorescently labeled nucleotides that were hybridizedwith the array after in situ synthesis of DNA oligos.

FIG. 14 is a photograph of a magnified portion of a top view of amicroarray having inulin as a reaction layer. The lighter spots arefluorescence of fluorescently labeled nucleotides that were hybridizedwith the array after in situ synthesis of DNA oligos.

FIG. 15 is a photograph of a magnified portion of a top view of amicroarray having palatinose as a reaction layer. The lighter spots arefluorescence of fluorescently labeled nucleotides that were hybridizedwith the array after in situ synthesis of DNA oligos.

FIG. 16 is a photograph of a magnified portion of a top view of amicroarray having polyethylene glycol as a reaction layer. The lighterspots are fluorescence of fluorescently labeled nucleotides that werehybridized with the array after in situ synthesis of DNA oligos.

FIG. 17 is a photograph of a magnified portion of a top view of amicroarray having salicin as a reaction layer. The lighter spots arefluorescence of fluorescently labeled nucleotides that were hybridizedwith the array after in situ synthesis of DNA oligos.

FIG. 18 is a photograph of a magnified portion of a top view of amicroarray having ribose as a reaction layer. The lighter spots arefluorescence of fluorescently labeled nucleotides that were hybridizedwith the array after in situ synthesis of DNA oligos.

FIG. 19 is a photograph of a magnified portion of a top view of amicroarray having melibiose as a reaction layer. The lighter spots arefluorescence of fluorescently labeled nucleotides that were hybridizedwith the array after in situ synthesis of DNA oligos.

FIG. 20A-20E are schematics of a cross-section of four electrodes of amicroarray of electrodes showing the synthesis of peptides on theelectrodes followed by exposure to anti-beta endorphin antibody (clone3-E7, mouse) and Cy5 labeled donkey anti-mouse antibody. Two electrodeshave an ionic linking group, and two electrodes are missing an ioniclinking group. Sequence listing: FIGS. 20B-30E, Elements 2016+2018(TTTTTTTTTTTTTTTT) (SEQ ID NO:3); FIGS. 20D-20E, Elements 2024 (Tyr GlyGly Phe Leu) (SEQ ID NO:2).

FIG. 21 is a schematic of a cross-section of two electrodes of amicroarray of electrodes showing the quenching of fluorescently labeledreagent by the platinum electrode when an ionic linker is not used.Sequence listing: Element 2116 (TTTTTTTTTTTTTTTT) (SEQ ID NO:3);Elements 2124 (Tyr Gly Gly Phe Leu) (SEQ ID NO:2).

FIG. 22 is a magnified and contrast-enhanced photograph of the top viewof a section of an electrode microarray showing that the fluorescence ofCy5 labeled donkey anti-mouse antibody is visible when an ionic linkeris used to connect the peptide to the platinum electrode overlayer.

FIG. 23 is schematic of a cross-section of two electrodes of amicroarray of electrodes. One electrode shows that the use of anon-ionic linker allows quenching of the Cy5 labeled donkey anti-mouseantibody because the non-ionic linker is poorly solvated. The otherelectrode shows that the use of an ionic linker prevents quenching ofthe Cy5 labeled donkey anti-mouse antibody because the ionic linker iswell solvated and thus keeps the labeled antibody away from the platinumelectrode. Sequence listing: Element 2316 (TTTTTTTTTTTTTTTT) (SEQ IDNO:3); Elements 2324 (Tyr Gly Gly Phe Leu) (SEQ ID NO:2).

FIG. 24 is a magnified photograph of the top view of a section of anelectrode microarray showing the decrease in fluorescence quenching asthe length of the linker/spacer is increased from 0 to 15deoxythymidylate units. The linker/spacer was synthesized in situ. Thefluorescence is from Texas Red labeled streptavidin bound to a biotinthat is covalently attached to the end of the linker/spacer.

FIG. 25 is a photograph of a magnified portion of a top view of amicroarray having 1-(3-hydroxylpropyl) pyrrole as a reaction layer. Thelighter spots are fluorescence of fluorescently labeled nucleotides thatwere hybridized with the array after in situ synthesis of DNA oligos.

FIG. 26 is a photograph of a magnified portion of a top view of amicroarray having 1-hexylpyrrole as a reaction layer. The lighter spotsare fluorescence of fluorescently labeled nucleotides that werehybridized with the array after in situ synthesis of DNA oligos.

FIG. 27 is a photograph of a magnified portion of a top view of amicroarray having a combination reaction layer, wherein the combinationcomprises sucrose and fructose. The lighter spots are fluorescence offluorescently labeled nucleotides that were hybridized with the arrayafter in situ synthesis of DNA oligos. The oligomers have a length of 35to 70 mers.

FIG. 28 is a photograph of a magnified portion of a top view of amicroarray having a combination reaction layer, wherein the combinationcomprises sucrose and fructose. The lighter spots are fluorescence offluorescently labeled nucleotides that were hybridized with the arrayafter in situ synthesis of DNA oligos. The oligomers have a length of 35mers.

FIG. 29 is a photograph of a magnified portion of a top view of amicroarray having a combination reaction layer, wherein the combinationcomprises sucrose and fructose. The lighter spots are fluorescence offluorescently labeled nucleotides that were hybridized with the arrayafter in situ synthesis of DNA oligos. The oligomers have an averagelength of 40 mers.

FIG. 30 is a photograph of a magnified portion of a top view of amicroarray having a combination reaction layer, wherein the combinationcomprises sucrose and fructose. The lighter spots are fluorescence offluorescently labeled nucleotides that were hybridized with the arrayafter in situ synthesis of DNA oligos. The oligomers have a length of 45mers.

FIG. 31 is a photograph of a magnified portion of a top view of amicroarray having a combination reaction layer, wherein the combinationcomprises sucrose and fructose. The lighter spots are fluorescence offluorescently labeled nucleotides that were hybridized with the arrayafter in situ synthesis of DNA oligos. The oligomers have a length of 50mers.

FIG. 32 is a photograph of a magnified portion of a top view of amicroarray having a combination reaction layer, wherein the combinationcomprises sucrose and fructose. The lighter spots are fluorescence offluorescently labeled nucleotides that were hybridized with the arrayafter in situ synthesis of DNA oligos. The oligomers have a length of 55mers.

FIG. 33 is a photograph of a magnified portion of a top view of amicroarray having a combination reaction layer, wherein the combinationcomprises sucrose and fructose. The lighter spots are fluorescence offluorescently labeled nucleotides that were hybridized with the arrayafter in situ synthesis of DNA oligos. The oligomers have a length of 60mers.

FIG. 34 is a photograph of a magnified portion of a top view of amicroarray having a combination reaction layer, wherein the combinationcomprises sucrose and fructose. The lighter spots are fluorescence offluorescently labeled nucleotides that were hybridized with the arrayafter in situ synthesis of DNA oligos. The oligomers have a length of 65mers.

FIG. 35 is a photograph of a magnified portion of a top view of amicroarray having a combination reaction layer, wherein the combinationcomprises sucrose and fructose. The lighter spots are fluorescence offluorescently labeled nucleotides that were hybridized with the arrayafter in situ synthesis of DNA oligos. The oligomers have a length of 70mers.

DETAILED DESCRIPTION OF THE INVENTION

For the most part, nomenclature for chemical groups as used hereinfollows the recommendations of “The International Union for Pure andApplied Chemistry”, Principles of Chemical Nomenclature: a Guide toIUPAC Recommendations, Leigh, G. J.; Favre, H. A. and Metanomski, W. V.,Blackwell Science, 1998, the disclosure of which is incorporated byreference herein. Formation of substituted structures is limited by atomvalence requirements.

“Oligomer” means a molecule of intermediate relative molecular mass, thestructure of which essentially comprises a small plurality of unitsderived, actually or conceptually, from molecules of lower relativemolecular mass. A molecule is regarded as having an intermediaterelative molecular mass if it has properties which do vary significantlywith the removal of one or a few of the units. If a part or the whole ofthe molecule has an intermediate relative molecular mass and essentiallycomprises a small plurality of units derived, actually or conceptually,from molecules of lower relative molecular mass, it may be described asoligomeric, or by oligomer used adjectivally. Oligomers are typicallycomprised of a monomer.

The term “co-oligomer” means an oligomer derived from more than onespecies of monomer. The term oligomer includes co-oligomers. As examplesof oligomers, a single stranded DNA molecule consisting ofdeoxyadenylate (A), deoxyguanylate (G), deoxycytidylate (C), anddeoxythymidylate (T) units in the following sequence, AGCTGCTATA (SEQ IDNO:1) is a co-oligomer, and a single stranded DNA molecule consisting of10-T units is an oligomer; however, both are referred to as oligomers.

The term “monomer” means a molecule that can undergo polymerizationthereby contributing constitutional units to the essential structure ofa macromolecule such as an oligomer, co-oligomer, polymer, orco-polymer. Examples of monomers include A, C, G, T, adenylate,guanylate, cytidylate, uridylate, amino acids, vinyl chloride, and othervinyls.

The term “polymer” means a substance composed of macromolecules, whichis a molecule of high relative molecular mass, the structure of whichessentially comprises the multiple repetition of units derived, actuallyor conceptually, from molecules of low relative molecular mass. In manycases, especially for synthetic polymers, a molecule can be regarded ashaving a high relative molecular mass if the addition or removal of oneor a few of the units has a negligible effect on the molecularproperties. This statement fails in the case of certain macromoleculesfor which the properties may be critically dependent on fine details ofthe molecular structure. If a part or the whole of the molecule has ahigh relative molecular mass and essentially comprises the multiplerepetition of units derived, actually or conceptually, from molecules oflow relative molecular mass, it may be described as eithermacromolecular or polymeric, or by polymer used adjectivally.

The term “copolymer” means a polymer derived from more than one speciesof monomer. Copolymers that are obtained by copolymerization of twomonomer species are sometimes termed bipolymers, those obtained fromthree monomers terpolymers, those obtained from four monomersquaterpolymers, etc. The term polymer includes co-polymers.

The term “polyethylene glycol” (PEG) means an organic chemical having achain consisting of the common repeating ethylene glycol unit[—CH₂—CH₂—O—]_(n). PEG's are typically long chain organic polymers thatare flexible, hydrophilic, enzymatically stable, and biologically inert,but they do not have an ionic charge in water. In general, PEG can bedivided into two categories. First, there is polymeric PEG having amolecular weight ranging from 1000 to greater than 20,000. Second, thereare PEG-like chains having a molecular weight that is less than 1000.Polymeric PEG has been used in bioconjugates, and numerous reviews havedescribed the attachment of this linker moiety to various molecules. PEGhas been used as a linker, where the short PEG-like linkers can beclassified into two types, the homo-[X—(CH₂—CH₂—O)_(n)]—X andheterobifunctional [X—(CH₂—CH₂—O)_(n)]—Y spacers.

The term “PEG derivative” means an ethylene glycol derivative having thecommon repeating unit of PEG. Examples of PEG derivatives include, butare not limited to, diethylene glycol (DEG), tetraethylene glycol (TEG),polyethylene glycol having primary amino groups, di(ethylene glycol)mono allyl ether, di(ethylene glycol) mono tosylate, tri(ethyleneglycol) mono allyl ether, tri(ethylene glycol) mono tosylate,tri(ethylene glycol) mono benzyl ether, tri(ethylene glycol) mono tritylether, tri(ethylene glycol) mono chloro mono methyl ether, tri(ethyleneglycol) mono tosyl mono allyl ether, tri(ethylene glycol) mono allylmono methyl ether, tetra(ethlyne glycol) mono allyl ether,tetra(ethylene glycol) mono methyl ether, tetra(ethylene glycol) monotosyl mono allyl ether, tetra(ethylene glycol) mono tosylate,tetra(ethylene glycol) mono benzyl ether, tetra(ethylene glycol) monotrityl ether, tetra(ethylene glycol) mono 1-hexenyl ether,tetra(ethylene glycol) mono 1-heptenyl ether, tetra(ethylene glycol)mono 1-octenyl ether, tetra(ethylene glycol) mono 1-decenyl ether,tetra(ethylene glycol) mono 1-undecenyl ether, penta(ethylene glycol)mono methyl ether, penta(ethylene glycol) mono allyl mono methyl ether,penta(ethylene glycol) mono tosyl mono methyl ether, penta(ethyleneglycol) mono tosyl mono allyl ether, hexa(ethylene glycol) mono allylether, hexa(ethylene glycol) mono methyl ether, hexa(ethylene glycol)mono benzyl ether, hexa(ethylene glycol) mono trityl ether,hexa(ethylene glycol) mono 1-hexenyl ether, hexa(ethylene glycol) mono1-heptenyl ether, hexa(ethylene glycol) mono 1-octenyl ether,hexa(ethylene glycol) mono 1-decenyl ether, hexa(ethylene glycol) mono1-undecenyl ether, hexa(ethylene glycol) mono 4-benzophenonyl mono1-undecenyl ether, hepta(ethylene glycol) mono allyl ether,hepta(ethylene glycol) mono methyl ether, hepta(ethylene glycol) monotosyl mono methyl ether, hepta(ethylene glycol) monoallyl mono methylether, octa(ethylene glycol) mono allyl ether, octa(ethylene glycol)mono tosylate, octa(ethylene glycol) mono tosyl mono allyl ether,undeca(ethylene glycol) mono methyl ether, undeca(ethylene glycol) monoallyl mono methyl ether, undeca(ethylene glycol) mono tosyl mono methylether, undeca(ethylene glycol) mono allyl ether, octadeca(ethyleneglycol) mono allyl ether, octa(ethylene glycol), deca(ethylene glycol),dodeca(ethylene glycol), tetradeca(ethylene glycol), hexadeca(ethyleneglycol), octadeca(ethylene glycol), benzophenone-4-hexa(ethylene glycol)allyl ether, benzophenone-4-hexa(ethylene glycol) hexenyl ether,benzophenone-4-hexa(ethylene glycol) octenyl ether,benzophenone-4-hexa(ethylene glycol) decenyl ether,benzophenone-4-hexa(ethylene glycol) undecenyl ether,4-flourobenzophenone-4′-hexa(ethylene glycol) allyl ether,4-flourobenzophenone-4′-hexa(ethylene glycol) undecenyl ether,4-hydroxybenzophenone-4′-hexa(ethylene glycol) allyl ether,4-hydroxybenzophenone-4′-hexa(ethylene glycol) undecenyl ether,4-hydroxybenzophenone-4′-tetra(ethylene glycol) allyl ether,4-hydroxybenzophenone-4′-tetra(ethylene glycol) undecenyl ether,4-morpholinobenzophenone-4′-hexa(ethylene glycol) allyl ether,4-morpholinobenzophenone-4′-hexa(ethylene glycol) undecenyl ether,4-morpholinobenzophenone-4′-tetra(ethylene glycol) allyl ether, and4-morpholinobenzophenone-4′-tetra(ethylene glycol) undecenyl ether.

The term “polyethylene glycol having primary amino groups” refers topolyethylene glycol having substituted primary amino groups in place ofthe hydroxyl groups. Substitution can be up to 98% in commercialproducts ranging in molecular weight from 5,000 to 20,000 Da.

The term “alkyl” means a straight or branched chain alkyl groupcontaining up to approximately 20 but preferably up to 8 carbon atoms.Examples of alkyl groups include but are not limited to the following:methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, isohexyl,n-hexyl, n-heptyl, and n-octyl. A substituted alkyl has one or morehydrogen atoms substituted by other groups or a carbon replaced by adivalent, trivalent, or tetravalent group or atom. Although alkyls bydefinition have a single radical, as used herein, alkyl includes groupsthat have more than one radical to meet valence requirements forsubstitution.

The term “alkenyl” means a straight or branched chain alkyl group havingat least one carbon-carbon double bond, and containing up toapproximately 20 but preferably up to 8 carbon atoms. Examples ofalkenyl groups include, but are not limited to, vinyl, 1-propenyl,2-butenyl, 1,3-butadienyl, 2-pentenyl, 2,4-hexadienyl,4-(ethyl)-1,3-hexadienyl, and 2-(methyl)-3-(propyl)-1,3-butadienyl. Asubstituted alkenyl has one or more hydrogen atoms substituted by othergroups or a carbon replaced by a divalent, trivalent, or tetravalentgroup or atom. Although alkenyls by definition have a single radical, asused herein, alkenyl includes groups that have more than one radical tomeet valence requirements for substitution.

The term “alkynyl” means a straight or branched chain alkyl group havinga single radical, having at least one carbon-carbon triple bond, andcontaining up to approximately 20 but preferably up to 8 carbon atoms.Examples of alkynyl groups include, but are not limited to, the ethynyl,1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 4-pentynyl,5-hexynyl, 6-heptynyl, 7-octynyl, 1-methyl-2-butynyl,2-methyl-3-pentynyl, 4-ethyl-2-pentynyl, and 5,5-methyl-1,3-hexynyl. Asubstituted alkynyl has one or more hydrogen atoms substituted by othergroups or a carbon replaced by a divalent, trivalent, or tetravalentgroup or atom. Although alkynyls by definition have a single radical, asused herein, alkynyl includes groups that have more than one radical tomeet valence requirements for substitution.

The term “cycloalkyl” means an alkyl group forming at least one ring,wherein the ring has approximately 3 to 14 carbon atoms. Examples ofcycloalkyl groups include but are not limited to the following:cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. A substitutedcycloalkyl has one or more hydrogen atoms substituted by other groups ora carbon replaced by a divalent, trivalent, or tetravalent group oratom. Although cycloalkyls by definition have a single radical, as usedherein, cycloalkyl includes groups that have more than one radical tomeet valence requirements for substitution.

The term “cycloalkenyl” means an alkenyl group forming at least one ringand having at least one carbon-carbon double bond within the ring,wherein the ring has approximately 3 to 14 carbon atoms. Examples ofcycloalkenyl groups include, but are not limited to, cyclopropenyl,cyclobutenyl, cyclopentenyl, 1,3-cyclopentadienyl, and cyclohexenyl. Asubstituted cycloalkenyl has one or more hydrogens substituted by othergroups or a carbon replaced by a divalent, trivalent, or tetravalentgroup or atom. Although cycloalkenyls by definition have a singleradical, as used herein, cycloalkenyl includes groups that have morethan one radical to meet valence requirements for substitution.

The term “cycloalkynyl” means an alkynyl group forming at least one ringand having at least one carbon-carbon triple bond, wherein the ringcontains up to approximately 14 carbon atoms. A group forming a ringhaving at least one triple bond and having at least one double bond is acycloalkynyl group. An example of a cycloalkynyl group includes, but isnot limited to, cyclooctyne. A substituted cycloalkynyl has one or morehydrogen atoms substituted by other groups. Although cycloalkynyls bydefinition have a single radical, as used herein, cycloalkynyl includesgroups that have more than one radical to meet valence requirements forsubstitution.

The term “aryl” means an aromatic carbon ring group having a singleradical and having approximately 4 to 20 carbon atoms. Examples of arylgroups include, but are not limited to, phenyl, naphthyl, and anthryl. Asubstituted aryl has one or more hydrogen atoms substituted by othergroups. Although aryls by definition have a single radical, as usedherein, aryl includes groups that have more than one radical to meetvalence requirements for substitution. An aryl group can be a part of afused ring structure such as N-hydroxysuccinimide bonded to phenyl(benzene) to form N-hydroxyphthalimide.

The term “hetero” when used in the context of chemical groups, or“heteroatom” means an atom other than carbon or hydrogen. Preferredexamples of heteroatoms include oxygen, nitrogen, phosphorous, sulfur,boron, silicon, and selenium.

The term “heterocyclic ring” means a ring structure having at least onering moiety having at least one heteroatom forming a part of the ring,wherein the heterocyclic ring has approximately 4 to 20 atoms connectedto form the ring structure. An example of a heterocyclic ring having 6atoms is pyridine with a single heteroatom. Additional examples ofheterocyclic ring structures having a single radical include, but arenot limited to, acridine, carbazole, chromene, imidazole, furan, indole,quinoline, and phosphinoline. Examples of heterocyclic ring structuresinclude, but are not limited to, aziridine, 1,3-dithiolane,1,3-diazetidine, and 1,4,2-oxazaphospholidine. Examples of heterocyclicring structures having a single radical include, but are not limited to,fused aromatic and non-aromatic structures: 2H-furo[3,2-b]pyran,5H-pyrido [2,3-d]-o-oxazine, 1H-pyrazolo[4,3-d]oxazole,4H-imidazo[4,5-d]thiazole, selenazolo [5,4-j]benzothiazole, andcyclopenta[b]pyran. Heterocyclic rings can have one or more radicals tomeet valence requirements for substitution.

The term “polycyclic” or “polycyclic group” means a carbon ringstructure having more than one ring, wherein the polycyclic group hasapproximately 4 to 20 carbons forming the ring structure and has asingle radical. Examples of polycyclic groups include, but are notlimited to, bicyclo[1.1.0]butane, bicyclo[5.2.0]nonane, andtricycle[5.3.1.1]dodecane. Polycyclic groups can have one or moreradicals to meet valence requirements for substitution. The term “halo”or “halogen” means fluorine, chlorine, bromine, or iodine.

The term “heteroatom group” means one heteroatom or more than oneheteroatoms bound together and having two free valences for forming acovalent bridge between two atoms. For example, the oxy radical, —O— canform a bridge between two methyls to form CH₃—O—CH₃ (dimethyl ether) orcan form a bridge between two carbons to form an epoxy such as cis ortrans 2,3-epoxybutane,

As used herein and in contrast to the normal usage, the term heteroatomgroup will be used to mean the replacement of groups in an alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl and not theformation of cyclic bridges, such as an epoxy, unless the term cyclicbridge is used with the term heteroatom group to denote the normalusage.

Examples of heteroatom groups, using the nomenclature for hetero bridges(such as an epoxy bridge), include but are not limited to the following:azimino (—N═N—HN—), azo (—N═N—), biimino (—NH—NH—), epidioxy epidithio(—S—S—), epithio (—S—), epithioximino (—S—O—NH—), epoxy (—O—),epoxyimino (—O—NH—), epoxynitrilo (—O—N═), epoxythio (—O—S—),epoxythioxy (—O—S—O—), furano (—C₄H₂O—), imino (—NH—), and nitrilo(—N═). Examples of heteroatom groups using the nomenclature for formingacyclic bridges include but are not limited to the following: epoxy(—O—), epithio (—S—), episeleno (—Se—), epidioxy (—O—O—), epidithio(—S—S—), lambda⁴-sulfano (—SH₂—), epoxythio (—O—S—), epoxythioxy(—O—S—O—), epoxyimino (—O—NH—), epimino (—NH—), diazano (—NH—NH—),diazeno (—N═N—), triaz[1]eno (—N═N—NH—), phosphano (—PH—), stannano(—SnH₂—), epoxymethano (—O—CH₂—), epoxyethano (—O—CH₂—CH₂—),epoxyprop[1]eno

The term “bridge” means a connection between one part of a ringstructure to another part of the ring structure by a hydrocarbon bridge.Examples of bridges include but are not limited to the following:methano, ethano, etheno, propano, butano, 2-buteno, and benzeno.

The term “hetero bridge” means a connection between one part of a ringstructure to another part of the ring structure by one or moreheteroatom groups, or a ring formed by a heterobridge connecting onepart of a linear structure to another part of the linear structure, thusforming a ring.

The term “oxy” means the divalent radical —O—.

The term “oxo” means the divalent radical ═O.

The term “carbonyl” means the group

wherein the carbon has two radicals for bonding.

The term “amide” or “acylamino” means the group

wherein the nitrogen has one single radical for bonding and R ishydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, orpolycyclic group.

The term “alkoxy” means the group —O—R, wherein the oxygen has a singleradical and R is hydrogen or an unsubstituted or substituted alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heterocyclic ring, or polycyclic group. Examples of alkoxy groups wherethe R is an alkyl include but are not limited to the following: methoxy,ethoxy, propoxy, butoxy, pentoxy, hexoxy, heptoxy, octoxy,1,1-dimethylethoxy, 1,1-dimethylpropoxy, 1,1-dimethylbutoxy,1,1-dimethylpentoxy, 1-ethyl-1-methylbutoxy, 2,2-dimethylpropoxy,2,2-dimethylbutoxy, 1-methyl-1-ethylpropoxy, 1,1-diethylpropoxy,1,1,2-trimethylpropoxy, 1,1,2-trimethylbutoxy,1,1,2,2-tetramethylpropoxy. Examples of alkoxy groups where the R is analkenyl group include but are not limited to the following: ethenyloxy,1-propenyloxy, 2-propenyloxy, 1-butenyloxy, 2-butenyloxy, 3-butenyloxy,1-methyl-prop-2-enyloxy, 1,1-dimethyl-prop-2-enyloxy,1,1,2-trimethyl-prop-2-enyloxy, and 1,1-dimethyl-but-2-enyloxy,2-ethyl-1,3-dimethyl-but-1-enyloxy. Examples of alkyloxy groups wherethe R is an alkynyl include but are not limited to the following:ethynyloxy, 1-propynyloxy, 2-propynyloxy, 1-butynyloxy, 2-butynyloxy,3-butynyloxy, 1-methyl-prop-2-ynyloxy, 1,1-dimethyl-prop-2-ynyloxy, and1,1-dimethyl-but-2-ynyloxy, 3-ethyl-3-methyl-but 1-ynyloxy. Examples ofalkoxy groups where the R is an aryl group include but are not limitedto the following: phenoxy, 2-naphthyloxy, and 1-anthyloxy.

The term “acyl” means the group

wherein the carbon has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup. Examples of acyl groups include but are not limited to thefollowing: acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl,acryloyl, propioloyl, mathacryloyl, crotonoyl, isocrotonoyl, benzoyl,and naphthoyl.

The term “acyloxy” means the group

wherein the oxygen has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup. Examples of acyloxy groups include but are not limited to thefollowing: acetoxy, ethylcarbonyloxy, 2-propenylcarbonyloxy,pentylcarbonyloxy, 1-hexynylcarbonyloxy, benzoyloxy,cyclohexylcarbonyloxy, 2-naphtho yloxy, 3-cyclodecenylcarbonyloxy.

The term “oxycarbonyl” means the group

wherein the carbon has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup. Examples of oxycarbonyl groups include but are not limited to thefollowing: methoxycarbonyl, ethoxycarbonyl, isopropyloxycarbonyl,phenoxycarbonyl, and cyclohexyloxycarbonyl.

The term “acyloxycarbonyl” means the group

wherein the carbon has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup.

The term “alkoxycarbonyloxy” means the group

wherein the oxygen has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup.

The term “carboxy” means the group —C(O)OH, wherein the carbon has asingle radical.

The term “imino” or “nitrene” means the group ═N—R, wherein the nitrogenhas two radicals and R is hydrogen or an unsubstituted or substitutedalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heterocyclic ring, or polycyclic group.

The term “amino” means the group —NH₂, where the nitrogen has a singleradical.

The term “secondary amino” means the group —NH—R, wherein the nitrogenhas a single radical and R is hydrogen or an unsubstituted orsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heterocyclic ring, or polycyclic group:

The term “tertiary amino” means the group

wherein the nitrogen has a single radical and R₁ and R₂ areindependently selected from the group consisting of unsubstituted andsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heterocyclic ring, and polycyclic group.

The term “hydrazi” means the group —NH—NH—, wherein the nitrogens havesingle radicals bound to the same atom. The term “hydrazo” means thegroup —NH—NH—, wherein the nitrogens have single radicals bound to thedifferent atoms.

The term “hydrazine” means the group NH₂—NH—, wherein the nitrogen has asingle radical.

The term “hydrazone” means the group NH₂—N═, wherein the nitrogen hastwo radicals.

The term “hydroxyimino” means the group HO—N═, wherein the nitrogen hastwo radicals.

The term “alkoxyimino” means the group R—O—N═, wherein the nitrogen hastwo radicals and R is an unsubstituted or substituted alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclicring, or polycyclic group.

The term “azido” means the group N₃—, wherein the nitrogen has oneradical.

The term “azoxy” means the group —N(O)═N—, wherein the nitrogens haveone radical.

The term “alkazoxy” means the group R—N(O)═N—, wherein the nitrogen hasone radical and R is hydrogen or an unsubstituted or substituted alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heterocyclic ring, or polycyclic group. Azoxybenzene is an examplecompound.

The term “cyano” means the group —CN. The term “isocyano” means thegroup —NC. The term “cyanato” means the group —OCN. The term“isocyanato” means the group —NCO. The term “fulminato” means the group—ONC. The term “thiocyanato” means the group —SCN. The term“isothiocyanato” means the group —NCS. The term “selenocyanato” meansthe group —SeCN. The term “isoselenocyanato” means the group —NCSe.

The term “carboxyamido” or “acylamino” means the group

wherein the nitrogen has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup.

The term “acylimino” means the group

wherein the nitrogen has two radicals and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup.

The term “nitroso” means the group O═N—, wherein the nitrogen has asingle radical.

The term “aminooxy” means the group —O—NH₂, wherein the oxygen has asingle radical.

The term “carxoimidioy” means the group

wherein the carbon has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup.

The term “hydrazonoyl” means the group

wherein the carbon has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup.

The term “hydroximoyl” or “oxime” means the group

wherein the carbon has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup.

The term “hydrazine” means the group

wherein the nitrogen has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup.

The term “amidino” means the group

wherein the carbon has a single radical.

The term “sulfide” means the group —S—R, wherein the sulfur has a singleradical and R is hydrogen or an unsubstituted or substituted alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heterocyclic ring, or polycyclic group.

The term “thiol” means the group —S—, wherein the sulfur has tworadicals. Hydrothiol means —SH.

The term “thioacyl” means the group —C(S)—R, wherein the carbon has asingle radical and R is hydrogen or an unsubstituted or substitutedalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heterocyclic ring, or polycyclic group.

The term “sulfoxide” means the group

wherein the sulfur has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup. The term “thiosulfoxide” means the substitution of sulfur foroxygen in sulfoxide; the term includes substitution for an oxygen-boundbetween the sulfur and the R group when the first carbon of the R grouphas been substituted by an oxy group and when the sulfoxide is bound toa sulfur atom on another group.

The term “sulfone” means the group

wherein the sulfur has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup. The term “thiosulfone” means substitution of sulfur for oxygen inone or two locations in sulfone; the term includes substitution for anoxygen bound between the sulfur and the R group when the first carbon ofthe R group has been substituted by an oxy group and when the sulfone isbound to a sulfur atom on another group.

The term “sulfate” means the group

wherein the oxygen has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup. The term “thiosulfate” means substitution of sulfur for oxygen inone, two, three, or four locations in sulfate.

The term “phosphoric acid ester” means the group R₁R₂PO₄—, wherein theoxygen has a single radical and R¹ is selected from the group consistingof hydrogen and unsubstituted and substituted alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, andpolycyclic group, and R² is selected from the group consisting ofunsubstituted and substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclicgroup.

The term “substituted” or “substitution,” in the context of chemicalspecies, means independently selected from the group consisting of (1)the replacement of a hydrogen on at least one carbon by a monovalentradical, (2) the replacement of two hydrogens on at least one carbon bya divalent radical, (3) the replacement of three hydrogens on at leastone terminal carbon (methyl group) by a trivalent radical, (4) thereplacement of at least one carbon and the associated hydrogens (e.g.,methylene group) by a divalent, trivalent, or tetravalent radical, and(5) combinations thereof. Meeting valence requirements restrictssubstitution. Substitution occurs on alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, andpolycyclic groups, providing substituted alkyl, substituted alkenyl,substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl,substituted cycloalkynyl, substituted aryl group, substitutedheterocyclic ring, and substituted polycyclic groups.

The groups that are substituted on an alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, andpolycyclic groups are independently selected from the group consistingof alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,aryl, heterocyclic ring, polycyclic group, halo, heteroatom group, oxy,oxo, carbonyl, amide, alkoxy, acyl, acyloxy, oxycarbonyl,acyloxycarbonyl, alkoxycarbonyloxy, carboxy, imino, amino, secondaryamino, tertiary amino, hydrazi, hydrazino, hydrazono, hydroxyimino,azido, azoxy, alkazoxy, cyano, isocyano, cyanato, isocyanato,thiocyanato, fulminato, isothiocyanato, isoselenocyanato, selenocyanato,carboxyamido, acylimino, nitroso, aminooxy, carboximidoyl, hydrazonoyl,oxime, acylhydrazino, amidino, sulfide, thiol, sulfoxide, thiosulfoxide,sulfone, thiosulfone, sulfate, thio sulfate, hydroxyl, formyl,hydroxyperoxy, hydroperoxy, peroxy acid, carbamoyl, trimethyl silyl,nitrilo, nitro, aci-nitro, nitroso, semicarbazono, oxamoyl, pentazolyl,seleno, thiooxi, sulfamoyl, sulfenamoyl, sulfeno, sulfinamoyl, sulfino,sulfinyl, sulfo, sulfoamino, sulfonato, sulfonyl, sulfonyldioxy,hydrothiol, tetrazolyl, thiocarbamoyl, thiocarbazono, thiocarbodiazono,thiocarbonohydrazido, thiocarbonyl, thiocarboxy, thiocyanato,thioformyl, thioacyl, thio semicarbazido, thio sulfino, thio sulfo,thioureido, thioxo, triazano, triazeno, triazinyl, trithio,trithiosulfo, sulfinimidic acid, sulfonimidic acid, sulfinohydrazonicacid, sulfonohydrazonic acid, sulfinohydroximic acid, sulfonohydroximicacid, and phosphoric acid ester, and combinations thereof.

As an example of a substitution, replacement of one hydrogen or ethaneby a hydroxyl provides ethanol, and replacement of two hydrogens by anoxo on the middle carbon of propane provides acetone (dimethyl ketone.)As a further example, replacement the middle carbon (the methenyl group)of propane by the oxy radical (—O—) provides dimethyl ether (CH₃—O—CH₃).As a further example, replacement of one hydrogen on a benzene by aphenyl group provides biphenyl. As provided above, heteroatom groups canbe substituted inside an alkyl, alkenyl, or alkylnyl group for amethylene group (:CH₂) thus forming a linear or branched substitutedstructure rather than a ring or can be substituted for a methyleneinside of a cycloalkyl, cycloalkenyl, or cycloalkynyl ring thus forminga heterocyclic ring. As a further example, nitrilo (—N═) can besubstituted on benzene for one of the carbons and associated hydrogen toprovide pyridine, or and oxy radical can be substituted to providepyran.

The term “unsubstituted” means that no hydrogen or carbon has beenreplaced on an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, or aryl group.

The term “linker” means a molecule having one end attached or capable ofattaching to a solid surface and the other end having a reactive groupthat is attached or capable of attaching to a chemical species ofinterest such as a small molecule, an oligomer, or a polymer. A linkermay already be bound to a solid surface and/or may already have achemical species of interest bound to its reactive group. A linker mayhave a protective group attached to its reactive group, where theprotective group is chemically or electrochemically removable. A linkermay comprise more than one molecule, where the molecules are covalentlyjoined in situ to form the linker having the desired reactive groupprojecting away from a solid surface.

The term “spacer” means a molecule having one end attached or capable ofattaching to the reactive group of a linker and the other end having areactive group that is attached or capable of attaching to a chemicalspecies of interest such as a small molecule, an oligomer, or a polymer.A spacer may already be bound to a linker and/or may already have achemical species of interest bound to its reactive group. A spacer mayhave a protective group attached to its reactive group, where theprotective group is chemically or electrochemically removable. A spacermay be formed in situ on a linker. A spacer may be formed and thenattached to a linker already attached to a solid surface. A spacer maybe externally synthesized on a chemical species of interest followed byattachment to a linker already attached to a solid surface. A chemicalspecies of interest may be attached to a spacer that is attached to alinker, where the entire structure is then attached to a solid surfaceat a reactive sight on the solid surface. The purpose of a spacer is toextend the distance between a molecule of interest and a solid surface.

The term “combination linker and spacer” means a linker having both theproperties of a linker and a spacer. A combination linker and spacer maybe synthesized in situ or synthesized externally and attached to a solidsurface.

The term “coating” means a thin layer of material that is chemicallyand/or physically bound to a solid surface. A coating may be attached toa solid surface by mechanical interlocking as well as by van der Waalsforces (dispersion forces and dipole forces), electron donor-acceptorinteractions, metallic coordination/complexation, covalent bonding, or acombination of the aforementioned. A coating can provide a reactivegroup for direct attachment of a chemical species of interest,attachment of a linker, or attachment of a combination linker andspacer. A coating can be polymerized and/or cross-linked in situ.

The term “reactive” or “reaction” as used in reactive or reactioncoating or reactive or reaction layer means that there is a chemicalspecies or bound group within the layer that is capable of forming acovalent bond for attachment of a linker, spacer, or other chemicalspecies to the layer or coating.

The term “porous” as used in porous reactive layer or coating means thatthere are non-uniformities within the layer or coating to allowmolecular species to diffuse into and through the layer or coating.

The term “adsorption” or “adsorbed” means a chemical attachment by vander Waals forces (dispersion forces and dipole forces), electrondonor-acceptor interactions, or metallic coordination/complexation, or acombination of the aforementioned forces. After adsorption, a speciesmay covalently bind to a surface, depending on the surface, the species,and the environmental conditions.

The term “microarray” refers to, in general, planer surface havingspecific spots that are usually arranged in a column and row format,wherein each spot can be used for some type of chemical or biochemicalanalysis, synthesis, or method. The spots on a microarray are typicallysmaller than 100 micrometers. The term “electrode microarray” refers toa microarray of electrodes, wherein the electrodes are the specificspots on the microarray.

The term “synthesis quality” refers to, in general, the average degreeof similarity between a desired or designed chemical or biochemicalspecies and the species actually synthesized. The term can refer toother issues in a synthesis such as the effect of a layer or coating onthe synthesis quality achieved.

The term “solvation” means a chemical process in which solvent moleculesand molecules or ions of a solute combine to form a compound, whereinthe compound is generally a loosely bound complex held together by vander Waals forces (dispersion forces and dipole forces), acid-baseinteractions (electron donor acceptor interactions), ionic interaction,or metal complex interactions but not covalent bonds. In water, the pHof the water can affect solvation of dissociable species such as acidsand bases. In addition, the concentration of salts as well as the chargeon salts can affect solvation.

The term “agarose” means any commercially available agarose. Agarose isa polysaccharide biopolymer and is usually obtained from seaweed.Agarose has a relatively large number of hydroxyl groups, which providefor high water solubility. Agarose is available commercially in a wideranger of molecular weights and properties.

The term “controlled pore glass” means any commercially availablecontrolled pore glass material suitable for coating purposes. Ingeneral, controlled pore glass (CPG) is an inorganic glass materialhaving a high surface area owing to a large amount of void space.

The term “monosaccharide” means one sugar molecule unlinked to any othersugars. Examples of monosaccharides include allose, altrose, arabinose,deoxyribose, erythrose, fructose (D-Levulose), galactose, glucose,gulose, idose, lyxose, mannose, psicose, ribose, ribulose,sedoheptulose, D-sorbitol, sorbose, sylulose, L-rhamnose(6-Deoxy-L-mannose), tagatose, talose, threose, xylulose, and xylose.

The term “disaccharide” means two sugars linked together to form onemolecule. Examples of disaccharides include amylose, cellobiose(4-.beta.-D-glucopyranosyl-D-glucopyranose), lactose, maltose(4-O-α-D-glucopyranosyl-D-glucose), melibiose(6-O-α-D-Galactopyranosyl-D-glucose), palatinose(6-O-α-D-Glucopyranosyl-D-fructose), sucrose, and trehalose(a-D-Glucopyranosyl-α-D-glucopyrano side).

The term “trisaccharide” means three sugars linked together to form onemolecule. Examples of a trisaccharides include raffinose(6-O-α-D-Galactopyranosyl-D-glucopyranosyl-.beta.-D-fructofuranoside)and melezitose(O-α-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-fructofuranosyl-α-D-glu-copyranoside).

The term “polysaccharide” means more than three sugars linked togetherto form one molecule, but more accurately means a sugar-based polymer oroligomer. Examples of polysaccharides include inulin, dextran (polymercomposed of glucose subunits), starches, and cellulose.

SPECIFIC EMBODIMENTS

In an embodiment of the present invention, an electrode microarrayhaving an adsorbed porous reaction layer for improved synthesis qualityis provided. The microarray has a plurality of electrodes attached to asubstrate, wherein the electrodes are electronically connected to acomputer control system that allows selection of any electrodeindividually or more than one electrode as group of electrodes. FIGS. 1Aand 1B are schematics of a cross section of two electrodes 108, 110 ofsuch a microarray 106 having a plurality of electrodes. In oneembodiment of the present invention, an adsorbed layer 104A shown inFIG. 1A covers the electrodes and the substrate that the electrodes areattached thereto. The adsorbed layer 104A has hydroxyl reactive groups102. The reactive groups 102 can be groups other than hydroxyl includingbut not limited to amine, carboxylic acid, aldehyde, thiol, alkene,alkyne, nitrile, azido, or phosphorous-based compound. In anotherembodiment shown in FIG. 1B, the adsorbed layer 104B can besubstantially on the electrodes but substantially not on the substrate106. In either embodiment, the adsorbed layer can be chemically blockedand selectively electrochemically deblocked to control the locations ofchemical reactions to specific electrodes while preventing chemicalreactions on non-selected electrodes and on non-electrode areas.

The adsorbed porous reaction layer on the plurality of electrodescomprises a chemical species having at least one hydroxyl group, whereinthe chemical species is selected from the group consisting ofmonosaccharides, disaccharides, trisaccharides, polyethylene glycol,polyethylene glycol derivative, N-hydroxysuccinimide, formula I, formulaII, formula III, formula IV, formula V, formula VI, and formula VII, andcombinations thereof, wherein formula I is

formula II is

formula III is HOR⁴(OR⁵)_(m)R⁷, formula IV is

formula V is

formula VI is

and formula VII is

wherein m is an integer from 1 to 4.

R¹, R², R⁷, and R⁸ are independently selected from the group consistingof hydrogen, and substituted and unsubstituted alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, andpolycyclic group, and halo, amide, alkoxy, acyl, acyloxy, oxycarbonyl,acyloxycarbonyl, alkoxycarbonyloxy, carboxy, amino, secondary amino,tertiary amino, hydrazino, azido, alkazoxy, cyano, isocyano, cyanato,isocyanato, thiocyanato, fulminato, isothiocyanato, isoselenocyanato,selenocyanato, carboxyamido, acylimino, nitroso, aminooxy,carboximidoyl, hydrazonoyl, oxime, acylhydrazino, amidino, sulfide,sulfoxide, thiosulfoxide, sulfone, thiosulfone, sulfate, thiosulfate,hydroxyl, formyl, hydroxyperoxy, hydroperoxy, peroxy acid, carbamoyl,trimethyl silyl, nitro, nitroso, oxamoyl, pentazolyl, sulfamoyl,sulfenamoyl, sulfeno, sulfinamoyl, sulfino, sulfo, sulfoamino,hydrothiol, tetrazolyl, thiocarbamoyl, thiocarbazono, thiocarbodiazono,thiocarbonohydrazido, thiocarboxy, thioformyl, thioacyl, thiocyanato,thio semicarbazido, thio sulfino, thio sulfo, thioureido, triazano,triazeno, triazinyl, trithiosulfo, sulfinimidic acid, sulfonimidic acid,sulfinohydrazonic acid, sulfonohydrazonic acid, sulfinohydroximic acid,sulfonohydroximic acid, and phosphoric acid ester;

R³ is preferably selected from the group consisting of heteroatom group,carbonyl, and substituted and unsubstituted alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, andpolycyclic group

R⁴ and R⁵ are preferably independently selected from the groupconsisting of methylene, ethylene, propylene, butylene, pentylene, andhexylene.

R⁶ forms a ring structure with two carbons of succinimide and isselected from the group consisting of substituted and unsubstitutedalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heterocyclic ring, and polycyclic group. R⁷ is selected from the groupconsisting of amino and hydroxyl.

Preferably, the monosaccharide is selected from the group consisting ofallose, altrose, arabinose, deoxyribose, erythrose, fructose(D-Levulose), galactose, glucose, gulose, idose, lyxose, mannose,psicose, L-rhamnose (6-Deoxy-L-mannose), ribose, ribulose,sedoheptulose, D-sorbitol, sorbose, sylulose, tagatose, talose, threose,xylulose, and xylose. Preferably, the disaccharide is selected from thegroup consisting of amylose, cellobiose(4-.beta.-D-glucopyranosyl-D-glucopyranose), lactose, maltose(4-O-α-D-glucopyranosyl-D-gluco se), melibiose(6-O-α-D-Galactopyranosyl-D-glucose), palatinose(6-O-α-D-Glucopyranosyl-D-fructose), sucrose, and trehalose(α-D-Glucopyranosyl-α-D-glucopyranoside). Preferably the triaccharide isselected from the group consisting of raffinose(6-O-α-D-Galactopyranosyl-D-glucopyranosyl-α-D-fructofuranoside) andmelezitose(O-α-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-fructofuranosyl-α-D-glucopyranoside).

Preferably the polyethylene glycol has a molecular weight betweenapproximately 1,000 and approximately 20,000, more preferably betweenapproximately 5000 and approximately 15,000, and most preferably betweenapproximately 7,000 and approximately 10,000.

Preferably, the polyethylene glycol derivative is selected from thegroup consisting of diethylene glycol, tetraethylene glycol,polyethylene glycol having primary amino groups, 2-(2-aminoethoxy)ethanol, ethanol amine, di(ethylene glycol) mono allyl ether,di(ethylene glycol) mono tosylate, tri(ethylene glycol) mono allylether, tri(ethylene glycol) mono tosylate, tri(ethylene glycol) monobenzyl ether, tri(ethylene glycol) mono trityl ether, tri(ethyleneglycol) mono chloro mono methyl ether, tri(ethylene glycol) mono tosylmono allyl ether, tri(ethylene glycol) mono allyl mono methyl ether,tetra(ethlyne glycol) mono allyl ether, tetra(ethylene glycol) monomethyl ether, tetra(ethylene glycol) mono tosyl mono allyl ether,tetra(ethylene glycol) mono tosylate, tetra(ethylene glycol) mono benzylether, tetra(ethylene glycol) mono trityl ether, tetra(ethylene glycol)mono 1-hexenyl ether, tetra(ethylene glycol) mono 1-heptenyl ether,tetra(ethylene glycol) mono 1-octenyl ether, tetra(ethylene glycol) mono1-decenyl ether, tetra(ethylene glycol) mono 1-undecenyl ether,penta(ethylene glycol) mono methyl ether, penta(ethylene glycol) monoallyl mono methyl ether, penta(ethylene glycol) mono tosyl mono methylether, penta(ethylene glycol) mono tosyl mono allyl ether, hexa(ethyleneglycol) mono allyl ether, hexa(ethylene glycol) mono methyl ether,hexa(ethylene glycol) mono benzyl ether, hexa(ethylene glycol) monotrityl ether, hexa(ethylene glycol) mono 1-hexenyl ether, hexa(ethyleneglycol) mono 1-heptenyl ether, hexa(ethylene glycol) mono 1-octenylether, hexa(ethylene glycol) mono 1-decenyl ether, hexa(ethylene glycol)mono 1-undecenyl ether, hexa(ethylene glycol) mono 4-benzophenonyl mono1-undecenyl ether, hepta(ethylene glycol) mono allyl ether,hepta(ethylene glycol) mono methyl ether, hepta(ethylene glycol) monotosyl mono methyl ether, hepta(ethylene glycol) monoallyl mono methylether, octa(ethylene glycol) mono allyl ether, octa(ethylene glycol)mono tosylate, octa(ethylene glycol) mono tosyl mono allyl ether,undeca(ethylene glycol) mono methyl ether, undeca(ethylene glycol) monoallyl mono methyl ether, undeca(ethylene glycol) mono tosyl mono methylether, undeca(ethylene glycol) mono allyl ether, octadeca(ethyleneglycol) mono allyl ether, octa(ethylene glycol), deca(ethylene glycol),dodeca(ethylene glycol), tetradeca(ethylene glycol), hexadeca(ethyleneglycol), octadeca(ethylene glycol), benzophenone-4-hexa(ethylene glycol)allyl ether, benzophenone-4-hexa(ethylene glycol) hexenyl ether,benzophenone-4-hexa(ethylene glycol) octenyl ether,benzophenone-4-hexa(ethylene glycol) decenyl ether,benzophenone-4-hexa(ethylene glycol) undecenyl ether,4-flourobenzophenone-4′-hexa(ethylene glycol) allyl ether,4-flourobenzophenone-4-hexa(ethylene glycol) undecenyl ether,4-hydroxybenzophenone-4′-hexa(ethylene glycol) allyl ether,4-hydroxybenzophenone-4′-hexa(ethylene glycol) undecenyl ether,4-hydroxybenzophenone-4′-tetra(ethylene glycol) allyl ether,4-hydroxybenzophenone-4′-tetra(ethylene glycol) undecenyl ether,4-morpholinobenzophenone-4′-hexa(ethylene glycol) allyl ether,4-morpholinobenzophenone-4′-hexa(ethylene glycol) undecenyl ether,4-morpholinobenzophenone-4′-tetra(ethylene glycol) allyl ether, and4-morpholinobenzophenone-4′-tetra(ethylene glycol) undecenyl ether.

More preferably, the adsorbed porous reaction layer chemical specieshaving at least one hydroxyl group is selected from the group consistingof sucrose, palatinose, fructose, glucose, lactose, DEG, TEG, and PEGhaving a molecular weight of approximately 8,000. Most preferably, theadsorbed porous reaction layer chemical species having at least onehydroxyl group is sucrose.

In an embodiment of the present invention, a method is provided forcleaning the electrode microarray prior to adsorption of the porousreaction layer onto the microarray. In one preferred embodiment forcleaning the microarray, the microarray is cleaned using a plasmacleaning method and then cleaned using an electrochemical cleaningmethod. In another preferred embodiment for cleaning the microarray, themicroarray is cleaned using a plasma cleaning method and then cleanedusing a hydrogen peroxide cleaning method.

In one embodiment of the present invention, the plurality of electrodeson the electrode microarray is selected from the group consisting ofplatinum, gold, semiconductor, indium tin oxide, and carbon, andcombinations thereof. Platinum is the preferred embodiment. In oneembodiment, the plasma cleaning method comprises exposing the electrodemicroarray to an inert gas to physically clean (sputter etch) thesurface of the electrode array. The inert gas is preferably Argon, andpreferably, the sputter etch process is performed for approximately twoto six minutes, wherein the plasma power is 200 W, the self bias voltageis 600-650V, the plasma pressure is 8 mTorr, and a 200 mm diameterelectrode is used in a parallel plate plasma chamber. In anotherembodiment, the plasma cleaning method comprises exposing the electrodemicroarray to a chemically reactive gas to clean the surface of theelectrode array through a chemical reactive process. The reactive gas ispreferably oxygen, sulfur hexafluoride, trifluoromethane, carbontetrafluoride or other chemically reactive gas species.

In a preferred embodiment the plasma process is performed in an OxfordInstruments Plasmalab 800 Plus RIE system having an electrode diameterof 460 mm, wherein the plasma cleaning method comprises a three-stepplasma treatment. In step one, the microarray is etched using argonplasma for approximately three minutes using a RF plasma power ofapproximately 600 watts, a set pressure of approximately eight millitorrand an Ar gas flow of approximately 30 sccm. In step two, the microarrayis etched using oxygen plasma for approximately six minutes using an RFplasma power of approximately 500 watts, a set pressure of approximately50 millitorr and an oxygen gas flow of approximately 50 sccm. For thefinal step, the microarray is etched using argon plasma forapproximately ten minutes using an RF plasma power of approximately 600watts, a set pressure of approximately eight millitorr and an Ar gasflow of approximately 30 sccm. Without being bound by the degree ofetching, the amount of material removed by etching is estimated to beapproximately equivalent to between 300 to 400 angstrom of a plasmaenhanced chemical vapor deposited (PECVD) silicon nitride film.

Preferably, the plasma cleaning method comprises exposing the electrodemicroarray to a sulfur hexafluoride plasma for approximately 30 to 60minutes, where the plasma power is 300 watts, the plasma pressure isapproximately 250 to 350 mTorr, and the gas flow is 124 cubiccentimeters per minute in an isoptropic plasma chamber. Preferably, theplasma cleaning method comprises etching the electrode microarray in acommercial Reactive Ion Etch Plasma system (such as Oxford Plasmalab800Plus RIE system with a 460 mm diameter electrode) using (1) an argonplasma for approximately 2 to 4 minutes and a RF plasma power ofapproximately 600 watts, where the pressure is approximately eightmillitorr and the Ar gas flow is approximately 30 sccm; (2) an oxygenplasma for approximately 5 to 7 minutes using a power of approximately500 watts, where the pressure is approximately 50 millitorr and theoxygen gas flow of approximately 50 sccm; or (3) an argon plasma forapproximately 8 to 12 minutes using a power of approximately 600 watts,where the pressure is approximately eight millitorr and the Ar gas flowis approximately 30 sccm.

In a preferred embodiment, the electrochemical cleaning method comprisesplacing the electrode microarray into a solution of sulfuric acid andthen pulsing columns of electrodes having an alternating pattern ofpulsed/active electrodes and ground electrodes. After the first pulse,the active electrodes become the ground electrodes and the groundelectrodes become the active electrodes. For each subsequent pulse, theelectrode columns alternate between being the active column and beingthe ground column. The electrode columns alternate between active andground for the duration of the cleaning time. The concentration ofsulfuric acid is between approximately 0.01 and 5 molar, more preferablybetween approximately 0.1 and 1.5 molar, and most preferably betweenapproximately 0.4 and 0.6 molar. The duration of the cleaning time isbetween approximately 1 and 60 minutes, more preferably betweenapproximately 5 and 15 minutes, and most preferably betweenapproximately 8 and 12 minutes. The pulse time (active electrode columntime) is between approximately 0.01 and 60 seconds, more preferablybetween approximately 0.05 and 0.5 seconds, and most preferably betweenapproximately 0.08-0.12 seconds. The cleaning is done preferably betweenapproximately 0.degree. C. and 50.degree. C. and most preferably betweenapproximately room temperature and 30.degree. C. After exposure to thesulfuric acid and electrical pulsing, the microarray is washed usingdistilled water. In one preferred embodiment, the concentration ofsulfuric acid is 0.5 molar, the cleaning time is 10 minutes, the pulsetime is 0.1 seconds, and the temperature is room temperature.

In a preferred embodiment, the hydrogen peroxide cleaning methodcomprises placing the electrode microarray into a solution containinghydrogen peroxide. The concentration of hydrogen peroxide is betweenapproximately 0.5 and 10 percent hydrogen peroxide, more preferablybetween approximately 1 and 5 percent hydrogen peroxide, and mostpreferably between approximately 2 and 4 percent hydrogen peroxide. Thetemperature of the solution is preferably between approximately roomtemperature and 95.degree. C., more preferably between approximately35.degree. C. and 80.degree. C., and most preferably betweenapproximately 60.degree. C. and 70.degree. C. The time of treatment ispreferably between approximately 1 minute and 24 hours, more preferablybetween approximately 30 minutes and 12 hours, and most preferablybetween approximately 45 minutes and 2 hours. In the most preferredembodiment, the concentration of hydrogen peroxide is 3 percent; thetreatment time is one hour; and the temperature of the solution is65.degree. C. Afterward exposure to the hydrogen peroxide solution, themicroarray is rinsed using distilled water.

In one embodiment of the present invention, an adsorption method isprovided for the attachment of the adsorbed porous reaction layerchemical species to a clean electrode microarray. The electrodemicroarray is placed into a solution containing a chemical species thatforms the reaction layer. Without being bound by theory, the chemicalspecies has a natural affinity for the clean microarray thus adsorbsonto the surface thereof. The treatment time is between approximately 1minute and 1 month, more preferably between approximately 30 minutes to1 week, and most preferably between approximately 1 hour and 24 hours.The solvent used for making the solution is preferably water. Othersolvents are suitable, including alcohols, acetonitrile, dimethylformamide and methylene chloride as well as other common laboratorysolvents or the uncommon equivalent of such solvents. Other unusualsolvents may be suitable. Any solvent that dissolves the chemicalspecies is suitable. The concentration of the chemical species insolution is between approximately 0.001 and 5 molar, more preferablybetween approximately 0.1 and 2 molar, and most preferably betweenapproximately 0.2 and 0.5 molar. The temperature of the solution duringtreatment is preferably between approximately 0 and 90.degree. C. In onepreferred embodiment, the solution is an aqueous solution of 0.25 molarsucrose; the treatment time is one hour; and the temperature is roomtemperature. In another preferred embodiment, the solution is an aqueoussolution of 0.25 molar sucrose; the treatment time is 48 hours; and thetemperature is 37.degree. C. After treatment, the microarray is rinsedusing the solvent used for the treatment solution. After rinsing, themicroarray is allowed to air dry.

In another embodiment of the present invention, an electrode microarrayhaving an adsorbed porous reaction layer having a combination linker andspacer (linker/spacer) attached thereto for improved synthesis qualityis provided. The microarray has a plurality of electrodes attached to asubstrate, wherein the electrodes are electronically connected to acomputer control system that allows selection of any electrodeindividually or more than one electrode as group of electrodes. In oneembodiment, the linker/spacer is synthesized in situ on the adsorbedporous reaction layer. In a preferred embodiment, the linker/spacer issynthesized in situ on an adsorbed porous reaction layer comprisingsucrose.

Embodiments of the present invention are provided in FIGS. 2A and 2B,which are schematics of a cross section of two electrodes 208, 210 of anelectrode microarray 206 having a plurality of electrodes. In oneembodiment, shown in FIG. 2A, the microarray 206 has an adsorbed porousreaction layer 204A having reacted hydroxyl groups 202 (shown afterreaction as an ether linkage) attached to the entire surface of themicroarray 206. A blocking group (P) 212 is shown attached to thereaction layer 204A at non-electrode locations. The blocking group 212prevents synthesis at non-electrode locations. In another embodiment,shown in FIG. 2B, the microarray 205 has an adsorbed porous reactionlayer 204B having reacted hydroxyl groups 202 (shown after reaction asan ether linkage), wherein the reaction layer is substantially attachedonly to electrodes 208, 210. In both embodiments, the electrodes 208,210 have a linker/spacer 214 attached thereto to the reaction layer204A, 204B. The linker/spacer 214 is attached through an ether linkage202. The linker/spacer has a terminal reactive group 216 for in situsynthesis.

In one embodiment of the present invention, the linker/spacer is anoligomer synthesized in situ and having a substantial charge in aqueoussolution. In a preferred embodiment, the linker/spacer is a sequence ofDNA synthesized in situ. FIG. 3 shows a cross section of two electrodes308, 310 of an electrode microarray 306 having a plurality ofelectrodes. An adsorbed porous reaction layer 304 is shown attached tothe microarray 306. Electrode 310 shows a DNA linker/spacer 316 having anegative charge 318 in an aqueous solution 320 having cations, whereinthe linker/spacer is attached to an adsorbed porous reaction layer 304through an ether linkage 302 and has a linked group 312 attached at theend of the linker/spacer 316 protruding into the aqueous solution 320.In a preferred embodiment, the linker/spacer comprises a 15-unitdeoxythymidylate DNA chain synthesized in situ. Electrode 308 shows anon-ionic linker/spacer 314 attached thereto via an ether linkage 302and having a linked group 312 terminally attached.

Without being bound by theory, the non-ionic linker/spacer 314 likelyallows the linked group 312 to approach the microarray 306 because thenon-ionic linker/spacer 314 is less well solvated owing to the lack ofionic charge. Without being bound by theory, the charge on thelinker/spacer 316 likely improves solvation in aqueous media 320 bypreventing the linker/spacer 316 from folding on itself because ofcharge repulsion. Charge repulsion prevents the linker/spacer 316 frominteracting with other adjacent charged linker/spacers on the sameelectrode. Additionally, salvation structures are likely formed in theaqueous media thus minimizing side chain contact of the chargedlinker/spacer with the solid surface. Without being bound by theory, awell solvated linker/spacer is expected to allow a subsequent groupplaced in a solution to have better access to the reactive group on theend of the linker/spacer while at the same time preventing quenching offluorescence from a fluorescently labeled marker attached to asubsequent group or a chain of subsequent groups.

The following examples are provided merely to explain, illustrate, andclarify the present invention and not to limit the scope or applicationof the present invention. One of skill in the art would readilyrecognize similar embodiments and applications of the present inventionthat fall within the scope of the present invention.

EXAMPLE 1

This example illustrates microarrays of nucleotides prepared usingselected adsorbed porous reaction layers on the different microarrays.Each microarray was cleaned using the plasma cleaning method and theelectrochemical cleaning method or using the plasma cleaning method andthe hydrogen peroxide cleaning method, each as disclosed herein. Aftercleaning, each microarray was exposed to a solution containing achemical for forming an adsorbed porous reaction layer as disclosedherein. The chemicals used for the experiments included agarose,sucrose, diethylene glycol, ethylene glycol, N-hydroxysuccinimide,triethylene glycol, raffinose, melizitose, Splenda®, inulin,polyethylene glycol having a molecular weight of 8000, salicin, ribose,and melibiose.

After each microarray was prepared, after cleaning with a porousreaction layer, different nucleotides of either 15 mer, shown in FIGS.11 though 19, 25, and 26, or 35 mer, shown in FIGS. 5 through 10 , weresynthesized in situ on each microarray using an electrochemicalsynthesis method. After synthesis, the 35 mer nucleotide microarrayswere hybridized to a complex background having a spiked-in controltranscript. The complex background sample was prepared fromfluorescently labeled placental DNA. The spiked-in control was a labeledphage lambda nucleic acid. Various amounts of spiked-in controltranscripts were combined with the complex background. The 15 mermicroarrays were hybridized to labeled 15 mer DNA oligonucleotides. The15 mer oligonucleotides were prepared from phage lambda nucleic acid.After hybridization to the spiked control transcripts in the complexbackground or the 15 mer labeled oligonucleotide, each microarray wasimaged to view the amount of fluorescence and the quality of thefluorescence on the microelectrodes of the microarrays. Quality wasconsidered “good” when there was a fluorescent circle on an electrodehaving a relatively uniform amount of fluorescence and having a sharploss of fluorescence at the edge of the circle. In addition, anotherquality parameter was where there was minimal fluorescence at locationsother than electrodes.

In FIGS. 27 through 35 , sucrose was blended with other saccharides toform an adsorbed porous reaction layer. The solution having the blendcontained 50 mM of sucrose, 100 mM of fructose, and 100 mM of glucose.Oligonucleotides were synthesized with lengths of 35, 40, 45, 50, 55,60, 65, and 70 mers on different quadrants of the microarray. FIG. 27shows a section of a microarray having each of the different length ofDNA mers. FIGS. 28 through 35 show a larger magnification of each of thequadrants in FIG. 27 . Synthesis quality was assessed by hybridizationof a labeled random 9 mer (sequence: NNN NNN NNN, where N=A, G, C, orT). Without being bound by theory, a blend of monosaccharides anddisaccharides used to form the adsorbed porous reaction layer ishypothesized to decrease the amount of DNA that is synthesized at eachelectrode. Furthermore, without being bound by theory, as the length ofthe oligo is increased, thus increasing the quantity of DNA made perspot, the DNA may be susceptible to sheer forces and may be coming offthe electrode when sucrose is used by itself. Glucose and fructoseprovided lower density of DNA synthesis at each electrode. Thus,blending sucrose with glucose and fructose is thought to reduce theamount of DNA per electrode. Using a blend as provided in the presentinvention, the amount, as well as the spacing, between the DNAsynthesized at each electrode is better controlled.

FIGS. 4 through 19, 25, 26, and 27 are magnified photographs of a topview of a portion of each microarray having a different adsorbed porousreaction layer. In FIG. 4 , the reaction layer was agarose. Electrode402 shows non-uniform fluorescence that indicated the synthesis was oflow quality. The low quality may have been a result of a separation ofthe agarose away from the electrode. The synthesis was performed in acheckerboard pattern of on and off electrodes where electrode 404 was anelectrode that was off. This result indicated that agarose is not thatsuitable for use as a reaction layer.

In FIG. 5 , the reaction layer was sucrose. Electrode 502 shows gooduniformity across the electrodes of the fluorescence, which indicated ahigh quality synthesis and a stable reaction layer. At electrode 504,there is some amount of spotting; however, on the whole, sucrose workedwell as a reaction layer.

FIG. 6 shows the results of a sensitivity study on a sucrose reactionlayer. Hybridization was done with and without transcript spike.Controls were done to ensure that the microarray was performing thesynthesis as designed. Comparing the results of spiked to non-spikedsamples, the microarray showed a sensitivity of approximately onepicomolar when sucrose is used as the porous reaction layer.

In FIG. 7 , the reaction layer is diethylene glycol. Electrodes 704,706, 708 show uniformity problems and loss of sharpness at the edge ofthe electrodes. Moreover, there were considerable random spotting 702.In FIG. 8 , the reaction layer is ethylene glycol. Electrodes 802 and804 showed some indication of synthesis; however, the overall qualitywas very low owing to the lack of synthesis and considerable amount ofrandom spotting. In FIG. 9 , the reaction layer is N-hydroxysuccinamide.Electrode 904 showed acceptable uniformity and sharpness at the edge ofthe electrode. However, electrodes 902 and 906 showed some randomspotting. In FIG. 10 , the reaction layer is triethylene glycol.Electrode 1002 showed good quality. Electrode 1004 showed some spotting.Electrode 1006 showed some halo effect where the middle part of theelectrode showed a loss of fluorescence, which may indicate a loss ofreaction layer.

In FIG. 11 , the reaction layer is raffinose. Although synthesisoccurred, electrodes 1102, 1104, and 1106 showed a fair amount ofnon-uniformity and random spotting. In FIG. 12 , the reaction layer ismelizitose. There was little, if any, indication of synthesis atelectrode 1202. There was some random spotting. In FIG. 13 , thereaction layer is Splenda®, a modified sucrose. Uniformity was fairlygood as shown at electrodes 1302 and 1304. Electrode 1306 showed somenon-uniformity and random spotting. In FIG. 14 , the reaction layer isinulin, a fructose oligomer. Electrodes 1402 and 1404 indicated spottingsynthesis. There was considerable random spotting as indicated byfeature 1406.

In FIG. 15 , the reaction layer is palatinose. Electrodes 1502 and 1504showed good uniformity and edge sharpness. Feature 1506 showed thatthere is some random spotting. In FIG. 16 , the reaction layer ispolyethylene glycol having a molecular weight of approximately 8000daltons. Electrodes 1602 and 1604 showed very good uniformity and edgesharpness. Additionally, there was minimal random spotting. In FIG. 17 ,the reaction layer is salicin. Electrodes 1702 and 1704 showed veryspotty and non-uniform synthesis. In FIG. 18 , the reaction layer isribose. Electrodes 1802 and 1804 showed minimal spotty synthesis. Therewas some random spotting. In FIG. 19 , the reaction layer is melibiose.Electrodes 1902 and 1904 showed non-uniform synthesis.

In FIG. 25 , the reaction layer is 1-(3-hydroxylpropyl) pyrrole. Theelectrodes showed a fairly uniform synthesis, indicating1-(3-hydroxylpropyl) pyrrole as a good candidate for uses as an adsorbedporous reaction layer. In FIG. 26 , the reaction layer is1-hexylpyrrole. The electrodes showed a fairly uniform synthesis,indicating 1-hexylpyrrole as a good candidate for uses as an adsorbedporous reaction layer. FIG. 27 is a photograph of a magnified portion ofa top view of a microarray having a combination reaction layer, whereinthe combination comprises sucrose, fructose, and glucose. The lighterspots are fluorescence of fluorescently labeled nucleotides that werehybridized with the array after in situ synthesis of DNA oligos. Theoligomers had a length of 35 to 70 mers. FIGS. 28 through 35 show eachquadrant of FIG. 27 .

EXAMPLE 2

This example illustrates a peptide array with and without a combinationlinker and spacer that was synthesized on an electrode microarray ofplatinum electrodes having an absorbed porous reaction layer comprisingsucrose. The combination linker and spacer was a 16 T unit synthesizedin situ. After synthesis of the combination linker and spacer, thepeptide array was synthesized in situ thereon. Fluorescent reagent wasused to image the peptides, but the only image that could be seen was onthe electrodes having the combination linker and spacer.

The electrode microarray used was a commercial microarray made byCombiMatrix Corporation (CUSTOMARRAY) (Dill et al., Anal. Chim. Acta2001, 444:69, and Montgomery I, II, and III). The microarray consistedof a semiconductor silicon chip with an array of 1024 individuallyserially addressable 92-micrometer diameter platinum electrodes in a16.times.64 pattern. Prior to using the microarray, the electrodes werecoated with sucrose to allow covalent bonding of chemical species to theelectrode via the adsorbed sucrose. The sucrose was adsorbed by exposingthe platinum electrodes to a solution of sucrose in water followed by awater rinse to remove excess sucrose. The electrodes were set to aspecified voltage via connection to a personal computer havingappropriate control software. The software allowed control of eachelectrode on the microarray for electrochemical deblocking in thesequential synthesis of small molecules, oligomers, and polymers,including oligos and peptides.

On four electrodes (FIG. 22 ), a 15-unit deoxythymidylate strand wassynthesized using standard phosphoramidite chemistry. For theelectrochemical deblocking steps, 1.8 volts was applied for 60 secondsusing an acetonitrile/methanol deblocking solution. Following thedeblocking of the 15^(th) deoxythymidylate, a deoxythymidylate having a5′ aminoethoxyethyl modifier was attached to electrodes having the 15deoxythymidylate units and attached to electrodes not having the 15deoxythymidylate units (FIG. 22 ). A modified deoxythymidylate can beobtained from Glen Research, Inc. The microarray was then fullydeprotected using standard chemical deblocking instead ofelectrochemical deblocking.

Chemical deblocking was accomplished by exposing the microarray to a oneto one solution of ethylene diamine and ethanol for one hour at65.degree. C. and then exposing to Deblock™ (Burdick and Jackson) for 30minutes at room temperature. Leucine (L) was coupled to eightelectrodes. The microarray was exposed to a solution containing t-BOCprotected L (120 milligrams, 0.52 millimoles),O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate(190 milligrams, 0.50 millimoles)(HBTU), N-hydroxybenztriazole (67milligrams, 0.50 millimoles)(HOBT), and diisopropylethylamine (261microliter, 1.50 millimoles)(DIPEA) dissolved into one milliliter ofN,N-dimethylformamide (DMF).

Following coupling of the leucine, the microarray was washedsuccessively with DMF (one milliliter) and then methylene chloride (onemilliliter). Following washing, the leucine-coupling step was repeatedto ensure complete coverage of the electrodes with the t-BOC protectedleucine. Following the second L couple step, the microarray was washedsuccessively with DMF (one milliliter), methylene chloride (onemilliliter), and then with ethanol to remove any residual DMF ormethylene chloride. The microarray was then allowed to air dry.

Following drying, the microarray was covered with a solution of1,2-diphenylhydrazine (200 milligrams, 1.1 millimoles) andtetrabutylammonium hexafluorophosphate (400 milligrams, 1.0 millimoles)dissolved in methylene chloride (10 milliliters). Using a computercontrol system, the eight selected electrodes were powered to make suchelectrodes the active electrodes to deblock the L on only the activeelectrodes. Deblocking removed the t-BOC protecting group from the L onthe active electrodes. The active electrodes were held at 3.0 voltsverses a platinum counter electrode for 60 seconds. After deblocking,the deblock solution was removed from the microarray. The microarray wasrinsed with ethanol to remove any residual electrochemical deblockingsolution and then allowed to air dry.

The synthesis process was repeated using a step pattern witht-BOC-phenylalanine-OH (F) (FIG. 22 ). Following this step,electrochemical deblocking was done. The synthesis process iteratedthrough two rounds of boc-glycine-OH (G) followed by Boc-tyrosine(t-butyl)-OH (Y) to construct a peptide having LFGGY as its sequence asviewed by moving away from the solid surface. The peptide sequence ismore commonly written as YGGFL (SEQ ID NO:2).

Once the microarray was constructed, instead of using electrochemicaldeblocking, the entire microarray was subjected to standard chemicaldeblocking using 40% trifluoroacetic acid (TFA) in methylene chloride(30 minutes) followed by 90% aqueous TFA (30 minutes). Followingdeblocking, the microarray was rinsed with ethanol and then blocked withacylated bovine serum albumin (ABSA) to eliminate background binding ofantibody. The solution used for blocking contained two milligrams permilliliter of ABSA in 2.times.PBS and 0.05% TWEEN 20™. The blockingreaction was allowed to proceed for 30 minutes. After blocking, themicroarray was incubated with primary anti-beta-endorphin antibody. Theantibody used was Clone 3-E7 (monoclonal, mouse) and was diluted by1/1000 using 2.times.PBS having 0.05% TWEEN 20™ therein. The 2.times.PBSand TWEEN 20™ were purchased from Chemicon International, Inc. Theanti-beta-endorphin antibody will selectively adsorb on the electrodeshaving the peptide sequence YGGFL (SEQ ID NO:2) synthesized thereon.Following incubation, the microarray was exposed to Cy5™ labeled donkeyanti-mouse antibody, which will selectively adsorb onto theanti-beta-endorphin antibody. The Cy5™ labeled donkey anti-mouseantibody was purchased from Integrated DNA Technologies. Finally, themicroarray was imaged on an Array Works® Imager to locate the electrodeshaving Cy5™ labeled donkey anti-mouse.

FIGS. 20A-20E are schematics of a cross-section of a microarray 2006 offour electrodes 2008, 2009, 2010, 2010 of a microarray of electrodes. Asequence of steps is shown for the synthesis of the combination linkerand spacer 2016, 2018, 2020A, 2020B on two electrodes 2010, 2011followed by peptide synthesis 2024 and labeling 2030, 2032, 2034. FIG.20A is a schematic of the electrode microarray 2006 before synthesis. Acoating 2004 is shown covering the microarray. The coating 2004 onlycovered the platinum electrodes 2008, 2009, 2010, 2011. The coating 2004is shown having hydroxyl groups as the reactive groups 2002A. Otherreactive groups may be used. For this experiment, the coating 2004 wasan adsorbed layer of sucrose; therefore, the reactive groups werehydroxyl groups. Step 2012 was a sequence of steps for the attachment ofthe combination linker and spacer to two electrodes 2010, 2011 in FIG.20B. Two electrodes 2008, 2009 are shown without the combination linkerand spacer but having the reactive group changed to an amine 2020A byattachment of a modified T 2014, 2018 according to the above procedure.Step 2022 was the first step in building the peptide 2024 by adding L tothe reactive amine groups 2020B as shown in FIG. 20C. Step 2026 wasmultiple steps for building the peptide chain 2024 as shown in FIG. 20Don all four electrodes 2008, 2009, 2010, 2011. Step 2028 is two stepsfor the adsorption of Clone 3-E7 antibody 2030 followed by adsorption ofthe Cy5™ labeled donkey anti-mouse 2032, 2034 according to the aboveprocedure as shown in FIG. 20E. The Cy5™ labeled donkey anti-mouse 2034on two electrodes 2008, 2009 is shown as shaded to indicate that thefluorescence is quenched by the platinum electrodes because the distancebetween the electrodes and the label is insufficient to preventquenching. In contrast, Cy5™ labeled donkey anti-mouse 2032 on twoelectrodes 2010, 2011 is shown as not shaded to indicate that thefluorescence is visible because the combination linker and spacerprovides sufficient distance between the label and the electrodes toprevent quenching. Although only one synthesis unit is shown perelectrode, there were actually many units at each electrode; however,for illustration purposes, only one unit is shown.

FIG. 21 is a schematic of two electrodes from FIG. 20E shown with andwithout the combination linker and spacer. FIG. 21 shows a cross sectionof the electrode microarray 2106 showing two electrodes 2108, 2110 andhaving a coating 2104 having reactive hydroxyl groups 2102. The coating2104 is sucrose and was present only on the platinum electrodes. FIG. 21shows the effect of the combination linker and spacer 2116 on thedistance between the Cy5 labeled donkey anti-mouse antibody 2132, 2134and the platinum electrodes 2108, 2110 and the accompanying effect onpreventing quenching. The fluorescence from the label 2134 on electrode2108 was quenched whereas the fluorescence from the label 2132 onelectrode 2110 was not quenched due to the further distance between theelectrode 2110 and the label 2132. Additionally, the T units 2114, 2116are shown have a negative charge 2136, which improved solvation inaqueous media 2140 having ions 2138. The negative counter ions are notshown. The ions shown are merely representative of any type of ion thatmay have been present in solution. The hydronium ion is shown torepresent acidic species as a result of the dissociation of thephosphate OH groups on the T units.

FIG. 22 is a magnified photograph of the eight electrodes used in thisexample. The four electrodes 2202 did not have the combination linkerand spacer and hence did not show any visible fluorescence from the Cy5labeled donkey anti-mouse antibody because of platinum quenching. Thefour electrodes 2204 did have the combination linker and spacer andhence did show the fluorescence from the Cy5 labeled donkey anti-mouseantibody.

EXAMPLE 3

An electrode microarray was prepared according to the procedures inExample 1 but with a series of linker/spacers of different lengths from0 to 15 T units. In addition, after the first amino acid, leucine, wasattached, no subsequent amino acids were attached. Instead, biotin wasattached to the leucine at the locations having the different lengths oflinker/spacers. Following attachment of the biotin, the microarray wascovered by a solution of Texas Red labeled streptavidin, whichselectively complexes to biotin. Image analysis was done on themicroarray to view the electrodes having the Texas Red labeledstreptavidin.

FIG. 24 is magnified photograph of a top view of a portion of themicroarray 2400 showing rows 2402, 2404, 2406, 2408, 2410, 2412, and2414. Moving from left to right, the length of the combination linkerand spacer was zero on the first electrode in rows 2402, 2408, and 2412.Moving from right to left, the length of the linker/spacer was zero onthe first electrode in rows 2403, 2410, and 2414. Row 2406 did not haveany synthesis thereon. For rows 2402, 2408, and 2412, the length of thelinker/spacer increased by one T unit moving from left to right. Forrows 2403, 2410, and 2414, the length of the combination linker andspacer increased by one T unit moving from right to left. To allow insitu synthesis, the cells without a T unit had one modified T unithaving the amine group according to example 1 and FIG. 21 , electrode2108. FIG. 24 shows that as the length of the combination linker andspacer increases, the fluorescence increases. At approximately 6 to 8 Tunits, the fluorescence began to increase substantially until reachingthe last cell having 15 T units having the strongest fluorescence. Thus,increasing the length of the linker/spacer can eliminate the quenchingeffect of the platinum electrode.

EXAMPLE 4

FIG. 23 is a schematic of a cross section of two cells 2308, 2310 of anelectrode microarray 2306 having a plurality of platinum electrodesserially and individually addressable. A coating 2304 having hydroxylreactive groups 2302 is shown. The coating 2304 was a sucrose layeradsorbed onto the platinum electrodes and was only present on theelectrodes. Electrode 2308 is shown with a non-ionic combination linkerand spacer 2314, and electrode 2310 is shown having a combination linkerand spacer 2316 in accordance with the present invention. The nonioniclinker 2314 was attached before or after synthesis of the combinationlinker and spacer 2316 in accordance with the present invention. Thepeptide 2324 was synthesized in situ on electrodes 2308 and 2310. Thefluorescent labeling procedure of examples 1 and 2 was used to label thepeptides 2330, 2332, 2334 at electrodes 2308 and 2310.

The nonionic combination linker and spacer 2314 was a PEG compound orother nonionic compound. Although PEG is water soluble, it is not aswell solvated as the multiple ionic combination linker and spacer 2316of the present invention because negative charges 2318 contributedsubstantially to solvation. The lack of charge on a PEG (or othernonionic) can allow the PEG to fold upon itself, to approach theelectrode surface, or to approach nearby PEG chains on the sameelectrode with the result that the fluorescence on electrode 2308 isexpected to be less than 2310 due to platinum quenching and due to lessaccess of the labeling species to the peptide. It is expected thatsynthesis efficiency will be higher using the multiple ionic combinationlinker and spacer 2316 of the present invention because of better accessto the reactive group 420A, 420B on the end of the combination linkerand spacer 2316.

What is claimed is:
 1. An electrode array comprising: (a) an electrodemicroarray comprising a plurality of electrodes on a substrate, whereeach of the plurality of electrodes is electronically connected to acomputer control system and where each electrode of the plurality ofelectrodes has a surface; (b) a porous reaction layer on the surface ofeach electrode of the plurality of electrodes, where the porous reactionlayer is formed from a chemical species dissolved in a solvent, wherethe chemical species is selected from the group consisting of one ormore monosaccharides and one or more disaccharides, where the chemicalspecies is not rinsed from the surface when the surface is rinsed withthe solvent alone, where the porous reaction layer is bound to thesurface; and (c) an oligonucleotide synthesized in situ on the porousreaction layer on the electrode array, where the oligonucleotide isbound to the porous reaction layer.
 2. The electrode array of claim 1,where the surface is made from one or more material selected from thegroup consisting of platinum, gold, semiconductor, indium tin oxide, andcarbon.
 3. The electrode array of claim 1, where the porous reactionlayer is bonded on the surface through one or more forces selected fromthe group consisting of Van der Waals, cohesion, adhesion and surfacetension.
 4. The electrode array of claim 1, where the one or moremonosaccharides are selected from the group consisting of allose,altrose, arabinose, deoxyribose, erythrose, fructose, galactose,glucose, gulose, idose, lyxose, mannose, psicose, L-rhamnose, ribose,ribulose, sedoheptulose, D-sorbitol, sorbose, sylulose, tagatose,talose, threose, xylulose, and xylose.
 5. The electrode array of claim1, where the one or more disaccharides are selected from the groupconsisting of amylose, cellobiose, lactose, maltose, melibiose,palatinose, and trehalose.
 6. The electrode array of claim 1, where thechemical species further comprises one or more trisaccharides.
 7. Theelectrode array of claim 6, where the one or more trisaccharides areselected from the group consisting of raffinose and melezitose.
 8. Theelectrode array of claim 1, where the chemical species is soluble in anaqueous solution.
 9. The electrode array of claim 1, where theoligonucleotide is at least partially synthesized using standardphosphoramidite chemistry and attached to a linker synthesized in situon the porous reaction layer.
 10. The electrode array of claim 9, wherethe oligonucleotide is between a 15 mer and a 70 mer.
 11. Theoligonucleotide of claim 1, where the oligonucleotide is between a 15mer and a 70 mer.
 12. An electrode array comprising: (a) an electrodemicroarray comprising a plurality of electrodes on a substrate, whereeach of the plurality of electrodes is electronically connected to acomputer control system and where each electrode of the plurality ofelectrodes has a surface; (b) a porous reaction layer on the surface ofeach electrode of the plurality of electrodes, where the porous reactionlayer is formed from a chemical species dissolved in a solvent, wherethe chemical species is selected from the group consisting of one ormore monosaccharides, and one or more disaccharides, where the chemicalspecies is not rinsed from the surface when the surface is rinsed withthe solvent alone; and (c) an oligonucleotide synthesized at leastpartially in situ on the porous reaction layer on the electrode array,where the oligonucleotide is bound to the porous reaction layer.
 13. Theelectrode array of claim 12, where the one or more monosaccharides areselected from the group consisting of allose, altrose, arabinose,deoxyribose, erythrose, fructose, galactose, glucose, gulose, idose,lyxose, mannose, psicose, L-rhamnose, ribose, ribulose, sedoheptulose,D-sorbitol, sorbose, sylulose, tagatose, talose, threose, xylulose, andxylose.
 14. The electrode array of claim 12, where the one or moredisaccharides are selected from the group consisting of amylose,cellobiose, lactose, maltose, melibiose, palatinose, and trehalosedisaccharide selected from the group consisting of amylose, cellobiose,lactose, maltose, melibiose, palatinose, and trehalose.
 15. Theelectrode array of claim 12, where the chemical species furthercomprises one or more trisaccharides.
 16. The electrode array of claim15, where the one or more trisaccharides are selected from the groupconsisting of raffinose and melezitose.
 17. The oligonucleotide of claim12, where the surface is one or more material selected from the groupconsisting of platinum, gold, semiconductor, indium tin oxide, andcarbon.
 18. The electrode array of claim 12, where the electrodemicroarray is etched with one or both a plasma cleaning method and achemical cleaning method prior to applying the porous reaction layer.19. The electrode array of claim 12, where the oligonucleotide issynthesized in situ with lengths between 15 mer and 70 mer.
 20. Theelectrode array of claim 12, further comprising where theoligonucleotide is synthesized partially externally and attached on theelectrode array.